Biomaterials (v.28, #16)

Plagiarism and redundancy by David Williams (2535).

RGD—Functionalized polymer brushes as substrates for the integrin specific adhesion of human umbilical vein endothelial cells by Stefano Tugulu; Paolo Silacci; Nikolaos Stergiopulos; Harm-Anton Klok (2536-2546).
This report demonstrates the feasibility of surface-initiated atom transfer radical polymerization to prepare thin polymer layers (“brushes”) that can be functionalized with short peptide ligands and which may be of use as coatings to promote endothelialization of blood-contacting biomaterials. The brushes are composed of poly(2-hydroxyethyl methacrylate) (PHEMA) or poly(poly(ethylene glycol) methacrylate) (PPEGMA), which do not only suppress non-specific adhesion of proteins and cells but also contain hydroxyl groups that can be used to introduce small peptide ligands. A protocol has been developed that allows functionalization of the brushes with RGD containing peptide ligands resulting in surface concentrations ranging from ∼0.5–12 pmol/cm2. At peptide surface concentrations >1–5.3 pmol/cm2, human umbilical vascular endothelial cells (HUVECs) were found to adhere and spread rapidly. A difference in size and morphology of focal adhesions between HUVECs immobilized on PHEMA and PPEGMA brushes was observed. It is proposed that this is due to the increased ethylene glycol spacer length and hydrophilicity of the PPEGMA brushes, which may lead to increased ligand mobility and reduced ligand–integrin affinity. HUVECs immobilized on the polymer brushes were also found to be able to retain homeostasis when exposed to shear stresses that simulated arterial blood flow.
Keywords: Surface-initiated atom transfer radical polymerization; Polymer brushes; Poly(poly(ethylene glycol) methacrylate); Poly (2-hydroxyethyl methacrylate); RGD peptide; Cell adhesion;

The influence of biomaterials on endothelial cell thrombogenicity by Alison P. McGuigan; Michael V. Sefton (2547-2571).
Driven by tissue engineering and regenerative medicine, endothelial cells are being used in combination with biomaterials in a number of applications for the purpose of improving blood compatibility and host integration. Endothelialized vascular grafts are beginning to be used clinically with some success in some centers, while endothelial seeding is being explored as a means of creating a vasculature within engineered tissues. The underlying assumption of this strategy is that when cultured on artificial biomaterials, a confluent layer of endothelial cells maintain their non-thrombogenic phenotype. In this review the existing knowledge base of endothelial cell thrombogenicity cultured on a number of different biomaterials is summarized. The importance of selecting appropriate endpoint measures that are most reflective of overall surface thrombogenicity is the focus of this review. Endothelial cells inhibit thrombosis through three interconnected regulatory systems (1) the coagulation cascade, (2) the cellular components of the blood such as leukocytes and platelets and (3) the complement cascade, and also through effects on fibrinolysis and vascular tone, the latter which influences blood flow. Thus, in order to demonstrate the thrombogenic benefit of seeding a biomaterial with EC, the conditions under which EC surfaces are more likely to exhibit lower thrombogenicity than unseeded biomaterial surfaces need to be consistent with the experimental context. The endpoints selected should be appropriate for the dominant thrombotic process that occurs under the given experimental conditions.
Keywords: Endothelial cells; Thrombogenicity;

Nanotoxicity of iron oxide nanoparticle internalization in growing neurons by Thomas R. Pisanic; Jennifer D. Blackwell; Veronica I. Shubayev; Rita R. Fiñones; Sungho Jin (2572-2581).
Magnetic nanoparticles (MNPs) have shown great promise for use as tools in a wide variety of biomedical applications, some of which require the delivery of large numbers of MNPs onto or into the cells of interest. Here we develop a quantifiable model cell system and show that intracellular delivery of even moderate levels of iron oxide (Fe2O3) nanoparticles may adversely affect cell function. More specifically, we show that exposure to increasing concentrations of anionic MNPs, from 0.15 to 15 mm of iron, results in a dose-dependent diminishing viability and capacity of PC12 cells to extend neurites in response to their putative biological cue, i.e. nerve growth factor. The cytotoxicity results of biomaterials in our model system imply that more study into the acute and long-term effects of cellular Fe2O3 internalization is both warranted and necessary.
Keywords: Nanoparticle; Magnetism; Neural cell; Cytotoxicity; Biocompatibility;

Bone formation at the surface of low modulus Ti–7.5Mo implants in rabbit femur by Dan-Jae Lin; Cheng-Chung Chuang; Jiin-Huey Chern Lin; Jing-Wei Lee; Chien-Ping Ju; Hsiang-Shu Yin (2582-2589).
The biocompatibility of the Ti–7.5Mo alloy was examined, because the alloy has a high-strength/modulus ratio and thus is a potential candidate for orthopedic applications. Cell viability assay using 3T3 cells revealed that the Ti–7.5Mo did not induce apparent cell death, when the cells were grown on disks made of the alloy or incubated with the alloy-conditioned medium at 37 or 72 °C for 24–72 h. The Ti–6Al–4V alloy was used as a control and did not cause apparent cell death either. Moreover, pins of 6 mm long and 2 mm in diameter of Ti–7.5Mo and Ti–6Al–4V were implanted into the left and right rabbit femurs, respectively, for 6, 12 and 26 weeks. New bone tissue grew to surround the pins, which spanned cortical and marrow regions, as shown by toluidine blue-stained bone sections of the three time points. Strikingly, the amount of new bone encircling the Ti–7.5Mo implant was approximate two-folds of that at Ti–6Al–4V by 26 weeks post-implantation. This facilitation of bone formation could be associated with the unique properties, such as a low modulus and the composition of Mo, of the Ti–7.5Mo.
Keywords: Cell viability; Implantation to femur; Bone growth; Bone marrow; Bioabsorption;

The differential influence of colocalized and segregated dual protein signals on neurite outgrowth on surfaces by Gerald N. Hodgkinson; Patrick A. Tresco; Vladimir Hlady (2590-2602).
We present an in vitro micropatterning approach in which the density and spatial presentation of two separate protein layers can be independently controlled to form cell stripe assays through (1) the simultaneous application of microcontact printing (μCP) and microfluidic network (μFN) patterning to generate alternating stripes of pure single protein layers or (2) through μCP onto a pre-adsorbed homogeneous protein layer to generate alternating single and dual protein stripes. This approach enabled the creation of choice boundaries in which protein–protein interactions were limited and the effects of spatially segregated or colocalized dual protein signals on model primary neuronal behavior could be readily interrogated and compared on both glass and tissue culture polystyrene substrates. Dorsal root ganglion (DRG) cell body attachment was dictated largely by non-specific cell adhesion interactions and interactions between the guidance molecules laminin and aggrecan were insufficient to explain aggrecan inhibition on neurite outgrowth. The presentation of a specific laminin epitope stabilized by interactions with aggrecan and destabilized by μCP was a strong predictor of neurite promoting activity. These observations provide evidence that aggrecan is intrinsically inhibitory and that laminin–aggrecan interactions do not diminish laminin growth promoting properties.
Keywords: Nerve regeneration; Micropatterning; Integrin; Laminin; Chondroitin sulfate; Cell adhesion;

Patterned PLG substrates for localized DNA delivery and directed neurite extension by Tiffany Houchin-Ray; Laura A. Swift; Jae-Hyung Jang; Lonnie D. Shea (2603-2611).
Tissue engineering strategies that enable nerve regeneration will require methods that can promote and direct neurite extension across the lesion. In this report, we investigate an in vitro combinatorial approach to directed neurite outgrowth using gene delivery from topographically patterned substrates, which can induce expression of neurotrophic factors to promote neurite extension and direct the extending neurites. Poly(lactide-co-glycolide) (PLG), which has been used to fabricate conduits or bridges for regeneration, was compression molded to create channels with 100, 150, and 250 μm widths. DNA complexes were immobilized to the PLG, and cells cultured on the substrate were transfected with efficiencies dependent on channel width and DNA amount. A co-culture model consisting of primary neurons and accessory cells was employed to investigate neurite outgrowth within the channels. Localized secretion of nerve growth factor (NGF) by the accessory cells promoted neuron survival and neurite extension. Neurons cultured in channels with NGF expression exhibited longer primary neurites than in the absence of channels. Neurons cultured in smaller width PLG microchannels exhibited a greater degree of directionality and less secondary sprouting than larger channels. Finally, surface immobilization allowed for the delivery of distinct plasmids from each channel, which may enable channels to be tailored for specific nerve tracts. This approach demonstrates the ability to combine gene delivery with physical guidance, and can be tailored to target specific axonal populations with varying neurotrophic factor requirements.
Keywords: Gene delivery; Substrate-mediated delivery; Nerve guidance; PLG; Nerve regeneration;

The effect of the microstructure of β-tricalcium phosphate on the metabolism of subsequently formed bone tissue by Takatoshi Okuda; Koji Ioku; Ikuho Yonezawa; Hideyuki Minagi; Giichiro Kawachi; Yoshinori Gonda; Hisashi Murayama; Yasuaki Shibata; Soichiro Minami; Shimeru Kamihira; Hisashi Kurosawa; Tohru Ikeda (2612-2621).
The response of bone cells to a newly developed porous β-tricalcium phosphate composed of rod-shaped particles (RSβ-TCP), β-TCP composed of conventional non-rod-shaped particles (Cβ-TCP), and hydroxyapatite (HA) was analyzed using in vivo implantation and in vitro osteoclastogenesis systems. Implantation of the materials into the rabbit femur showed that RSβ-TCP and Cβ-TCP were bioresorbable, but HA was not. Up to 12 weeks after the implantation, bioresorption of RSβ-TCP and Cβ-TCP accompanied by the formation of new bone occurred satisfactorily. At 24 weeks post-implantation, most of the RSβ-TCP had been absorbed, and active osteogenesis was preserved in the region. However, in the specimens implanted with Cβ-TCP, the amount of not only the implanted Cβ-TCP but also the newly formed bone tissue decreased, and bone marrow dominated the region. The implanted HA was unbioresorbable throughout the experimental period. When osteoclasts were generated on RSβ-TCP, Cβ-TCP, or HA disks, apparent resorption lacunae were formed on the RSβ-TCP and Cβ-TCP, but not HA disks. Quantitation of the calcium concentration in the culture media showed an earlier and more constant release of calcium from RSβ-TCP than Cβ-TCP. These results showed that the microstructure of β-TCP affects the activity of bone cells and subsequent bone replacement.
Keywords: Biodegradation; Bone graft; Coculture; Osteoblast; Osteoclast;

Suppression of apoptosis by enhanced protein adsorption on polymer/hydroxyapatite composite scaffolds by Kyung Mi Woo; Jihye Seo; Ruiyun Zhang; Peter X. Ma (2622-2630).
Bone tissue engineering is a promising alternative to bone grafting. Scaffolds play a critical role in tissue engineering. Composite scaffolds made of biodegradable polymers and bone mineral-like inorganic compounds have been reported to be advantageous over plain polymer scaffolds by our group and others. In this study, we compared cellular and molecular events during the early periods of osteoblastic cell culture on poly(l-lactic acid)/hydroxyapatite (PLLA/HAP) composite scaffolds with those on plain PLLA scaffolds, and showed that PLLA/HAP scaffolds improved cell survival over plain PLLA scaffolds. Most cells (MC3T3-E1) on PLLA/HAP scaffolds survived the early culture. In contrast, about 50% of the cells initially adhered to the plain PLLA scaffolds were detached within the first 12 h and showed characteristics of apoptotic cell death, which was confirmed by TUNEL staining and caspase-3 activation. To investigate the mechanisms, we examined the adsorption of serum protein and adhesion molecules to the scaffolds. The PLLA/HAP scaffold adsorbed more than 1.4 times of total serum protein and much greater amounts of serum fibronectin and vitronectin than pure PLLA scaffolds. Similarly, significantly larger amounts of individual adhesion proteins and peptides (fibronectin, vitronectin, RGD, and KRSR) were adsorbed on the PLLA/HAP scaffolds than on the PLLA scaffolds, which resulted in higher cell density on the PLLA/HAP scaffolds. Furthermore, β1 and β3 integrins and phosphorylation of Fak and Akt proteins in the cells on the PLLA/HAP scaffolds were significantly more abundent than those on PLLA scaffolds, which suggest that enhanced adsorption of serum adhesion proteins to PLLA/HAP scaffolds protect the cells from apoptosis possibly through the integrin–FAK–Akt pathway. These results demonstrate that biomimetic composite scaffolds are advantageous for bone tissue engineering.
Keywords: Composite scaffold; Osteoblast; Bone tissue engineering; Polymer; Hydroxyapatite; Protein adsorption;

Osteogenic differentiation of rabbit mesenchymal stem cells in thermo-reversible hydrogel constructs containing hydroxyapatite and bone morphogenic protein-2 (BMP-2) by Kun Na; Sung won Kim; Bo Kyung Sun; Dae Gyun Woo; Han Na Yang; Hyung Min Chung; Keun Hong Park (2631-2637).
The aim of this study was to assess the efficacy of ectopic bone formation in a three-dimensional hybrid scaffold in combination with hydroxyapatite (HA) and poly(NiPAAm-co-AAc) as an injectable vehicle in the form of a supporting matrix for the osteogenic differentiation of rabbit mesenchymal stem cells (MSCs). Osteogenic differentiation of MSCs in the hybrid scaffold was greatly influenced by the addition of growth factors. When the osteoinduction activity of hybrid scaffold was studied following implantation into the back subcutis of nude mouse in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds containing growth factor (BMP-2: bone morphogenic protein-2). The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture group. We conclude that combination of MSC-seeded hybrid scaffold containing BMP-2 was a promising method by which to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.
Keywords: Thermo-sensitive hydrogel; Hydroxyapatite; BMP-2; MSCs; Bone;

Biocompatibility of nanoporous alumina membranes for immunoisolation by Kristen E. La Flamme; Ketul C. Popat; Lara Leoni; Erica Markiewicz; Thomas J. La Tempa; Brian B. Roman; Craig A. Grimes; Tejal A. Desai (2638-2645).
Cellular immunoisolation using semi-permeable barriers has been investigated over the past several decades as a promising treatment approach for diseases such as Parkinson's, Alzheimer's, and Type 1 diabetes. Typically, polymeric membranes are used for immunoisolation applications; however, recent advances in technology have led to the development of more robust membranes that are able to more completely meet the requirements for a successful immunoisolation device, including well controlled pore size, chemical and mechanical stability, nonbiodegradability, and biocompatibility with both the graft tissue as well as the host. It has been shown previously that nanoporous alumina biocapsules can act effectively as immunoisolation devices, and support the viability and functionality of encapsulated β cells. The aim of this investigation was to assess the biocompatibility of the material with host tissue. The cytotoxicity of the capsule, as well as its ability to activate complement and inflammation was studied. Further, the effects of poly(ethylene glycol) (PEG) modification on the tissue response to implanted capsules were studied. Our results have shown that the device is nontoxic and does not induce significant complement activation. Further, in vivo work has demonstrated that implantation of these capsules into the peritoneal cavity of rats induces a transient inflammatory response, and that PEG is useful in minimizing the host response to the material.
Keywords: Bioartificial pancreas; Cell encapsulation; Alumina; Biocompatibility;

The effect of the controlled release of basic fibroblast growth factor from ionic gelatin-based hydrogels on angiogenesis in a murine critical limb ischemic model by Hans Layman; Maria-Grazia Spiga; Toby Brooks; Si Pham; Keith A. Webster; Fotios M. Andreopoulos (2646-2654).
The localized delivery of exogenous, angiogenic growth factors has become a promising alternative treatment of peripheral artery disease (PAD) and critical limb ischemia. In the present study, we describe the development of a novel controlled release vehicle to promote angiogenesis in a murine critical limb ischemic model. Ionic, gelatin-based hydrogels were prepared by the carbodiimide-mediated amidation reaction between the carboxyl groups of gelatin or poly-l-glutamic acid molecules and the amine groups of poly-l-lysine or gelatin molecules, respectively. The degree of swelling of the synthesized hydrogels was assessed as a function of EDC/NHS ratios and the pH of the equilibrating medium, while the release kinetic profile of basic fibroblast growth factor (FGF-2) was evaluated in human fibroblast cultures. The degree of swelling (DS) decreased from 26.5±1.7 to 18.5±2.4 as the EDC concentration varied from 0.75 to 2.5 mg/ml. Eighty percent of the FGF-2 was released at controlled rates from gelatin–polylysine (gelatin–PLL) and gelatin–polyglutamic acid (gelatin–PLG) hydrogel scaffolds over a period of 28 days. Cell adhesion studies revealed that the negatively charged surface of the gelatin–PLG hydrogels exhibited superior adhesion capabilities in comparison to gelatin–PLL and control gelatin surfaces. Laser Doppler perfusion imaging as well as CD31+ capillary immunostaining demonstrated that the controlled release of FGF-2 from ionic gelatin-based hydrogels is superior in promoting angiogenesis in comparison to the bolus administration of the growth factor. Over 4 weeks, FGF-2 releasing gelatin–PLG hydrogels exhibited marked reperfusion with a Doppler ratio of 0.889 (±0.04) which was 69.3% higher than in the control groups.
Keywords: Angiogenesis; Hydrogels; FGF-2; Controlled delivery; Critical limb ischemia;

Selective uptake of surface-modified phospholipid vesicles by bone marrow macrophages in vivo by Keitaro Sou; Beth Goins; Shinji Takeoka; Eishun Tsuchida; William T. Phillips (2655-2666).
An advantage of using vesicles (liposomes) as drug delivery carriers is that their pharmacokinetics can be controlled by surface characteristics, which can permit specific delivery of the encapsulated agents to organs or cells in vivo. Here we report a vesicle formulation which targets the bone marrow after intravenous injection in rabbits. Surface modification of the vesicle with an anionic amphiphile; l-glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA) results in significant targeting of vesicles to bone marrow. Further incorporation of as little as 0.6 mol% of poly(ethylene glycol)-lipid (PEG-DSPE) passively enhanced the distribution of SA-vesicles into bone marrow and inhibited hepatic uptake. In this model, more than 60% of the intravenously injected vesicles were distributed to bone marrow within 6 h after administration of a small dose of lipid (15 mg/kg b.w.). Histological evidence indicates that the targeting was achieved due to uptake by bone marrow macrophages (BMMφ). The efficient delivery of encapsulated scintigraphic and fluorescent imaging agents to BMMφ suggests that vesicles are promising carriers for the specific targeting of BMMφ and may be useful for delivering a wide range of therapeutic agents to bone marrow.
Keywords: Nanoparticle; Liposome; Bone marrow; Macrophage; Drug delivery; Surface modification;

The attenuation of experimental lung metastasis by a bile acid acylated-heparin derivative by Kyeongsoon Park; Seok Ki Lee; Dai Hyun Son; Soo Ah Park; Kwangmeyung Kim; Hyo Won Chang; Eun-jeong Jeong; Rang-Woon Park; In-San Kim; Ick Chan Kwon; Youngro Byun; Sang Yoon Kim (2667-2676).
The inhibitory efficacies of new bile acid acylated-heparin derivative (heparin-DOCA) were evaluated on experimental lung metastasis. We evaluated the effect of heparin-DOCA on intercellular interactions including those between B16F10 and thrombin-activated platelets and TNF-α-activated HUVECs, and between B16F10 and immobilized mouse P-selectin. In addition, the inhibitory effects of heparin-DOCA on adhesion and invasion of B16F10 to Matrigel were studied. In an animal mouse study, the blood clot formation and the retention of red fluorescence protein (RFP)-B16F10 in lungs were assessed after heparin-DOCA and RFP-B16F10 intravenous administration. Furthermore, we investigated the anti-metastatic effect of heparin-DOCA against lung metastasis induced by B16F10 and SCC7. Heparin-DOCA inhibited intercellular interactions between B16F10 and activated platelets or activated HUVECs by blocking P- and E-selectin-mediated interactions. Moreover, it reduced adhesion and invasion of B16F10 to ECM, thereby affecting the reduction of early retention of B16F10 in the lung. Heparin-DOCA attenuated lung colony formation on the surfaces and in interior of the lung, and attenuated metastasis by B16F10 and SCC7. These results suggest that heparin-DOCA may have potentials as therapeutic agent that prevents tumor metastasis and progression.
Keywords: Bile acid acylated-heparin derivative; B16F10 melanoma; Adhesion; Invasion; Lung metastasis;

The influence of BMP-2 and its mode of delivery on the osteoconductivity of implant surfaces during the early phase of osseointegration by Yuelian Liu; Lukas Enggist; Alexander F. Kuffer; Daniel Buser; Ernst B. Hunziker (2677-2686).
Osteogenic agents, such as bone morphogenetic protein-2 (BMP-2), can stimulate the degradation as well as the formation of bone. Hence, they could impair the osteoconductivity of functionalized implant surfaces.We assessed the effects of BMP-2 and its mode of delivery on the osteoconductivity of dental implants with either a naked titanium surface or a calcium-phosphate-coated one. The naked titanium surface bore adsorbed BMP-2, whilst the coated one bore incorporated, adsorbed, or incorporated and adsorbed BMP-2. The implants were inserted into the maxillae of adult miniature pigs. The volume of bone deposited within a defined “osteoconductive” (peri-implant) space, and bone coverage of the implant surface delimiting this space, were estimated morphometrically 1–3 weeks later.After 3 weeks, the volume of bone deposited within the osteoconductive space was highest for coated and uncoated implants bearing no BMP-2, followed by coated implants bearing incorporated BMP-2; it was lowest for coated implants bearing only adsorbed BMP-2. Bone-interface coverage was highest for coated implants bearing no BMP-2, followed by coated implants bearing either incorporated, or incorporated and adsorbed BMP-2; it was lowest for uncoated implants bearing adsorbed BMP-2. Hence, the osteoconductivity of implant surfaces can be significantly modulated by BMP-2 and its mode of delivery.
Keywords: Biomimetic material; Bone morphogenetic protein (BMP); Bone; Bone healing; Osteoconduction;

Encapsulation and release of a hydrophobic drug from hydroxyapatite coated liposomes by Qingguo Xu; Yasuhiro Tanaka; Jan T. Czernuszka (2687-2694).
Hydroxyapatite (HA) coated liposomes (HACL) have been successfully manufactured and filled with a model hydrophobic (lipophilic) drug, indomethacin (IMC). These HACL particles have been characterized in terms of particle size and ζ-potential. The liposomes are formed from 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA) and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Altering their relative proportions caused the ζ-potential to change from −38.8 to −67.0 mV, with a concomitant change in phase transition temperature from 36.4 to 53.3 °C. These changes also affect the drug loading efficiency.The release profiles of IMC have been measured. HA coating of the liposome reduces the release rate of IMC over uncoated liposomes. Under the present experimental conditions 70% of the drug is released after approximately 5 h from the liposome, but coating with HA changes this time to over 20 h. Perhaps most importantly, it has been observed that for uncoated liposomes, IMC is released at a greater rate at pH=7.4 than at pH=4. However, coating with HA reduced the rate at pH=7.4 compared to pH=4. This behaviour arises because IMC is more soluble under basic conditions, but HA is more soluble under acidic conditions. This behaviour shows that it is now possible to have environmental control over the release of drugs from HA-coated liposomes.
Keywords: Liposome; Hydroxyapatite; Indomethacin; Constant composition precipitation; Drug release;