Biomaterials (v.30, #12)

The challenge of establishing preclinical models for segmental bone defect research by Johannes C. Reichert; Siamak Saifzadeh; Martin E. Wullschleger; Devakara R. Epari; Michael A. Schütz; Georg N. Duda; Hanna Schell; Martijn van Griensven; Heinz Redl; Dietmar W. Hutmacher (2149-2163).
A considerable number of international research groups as well as commercial entities work on the development of new bone grafting materials, carriers, growth factors and specifically tissue-engineered constructs for bone regeneration. They are strongly interested in evaluating their concepts in highly reproducible large segmental defects in preclinical and large animal models. To allow comparison between different studies and their outcomes, it is essential that animal models, fixation devices, surgical procedures and methods of taking measurements are well standardized to produce reliable data pools and act as a base for further directions to orthopaedic and tissue engineering developments, specifically translation into the clinic. In this leading opinion paper, we aim to review and critically discuss the different large animal bone defect models reported in the literature. We conclude that most publications provide only rudimentary information on how to establish relevant preclinical segmental bone defects in large animals. Hence, we express our opinion on methodologies to establish preclinical critically sized, segmental bone defect models used in past research with reference to surgical techniques, fixation methods and postoperative management focusing on tibial fracture and segmental defect models.
Keywords: Bone defects; Bone engineering; Animal models; Preclinical studies; Orthopaedic research;

Organ printing: Tissue spheroids as building blocks by Vladimir Mironov; Richard P. Visconti; Vladimir Kasyanov; Gabor Forgacs; Christopher J. Drake; Roger R. Markwald (2164-2174).
Organ printing can be defined as layer-by-layer additive robotic biofabrication of three-dimensional functional living macrotissues and organ constructs using tissue spheroids as building blocks. The microtissues and tissue spheroids are living materials with certain measurable, evolving and potentially controllable composition, material and biological properties. Closely placed tissue spheroids undergo tissue fusion — a process that represents a fundamental biological and biophysical principle of developmental biology-inspired directed tissue self-assembly. It is possible to engineer small segments of an intraorgan branched vascular tree by using solid and lumenized vascular tissue spheroids. Organ printing could dramatically enhance and transform the field of tissue engineering by enabling large-scale industrial robotic biofabrication of living human organ constructs with “built-in” perfusable intraorgan branched vascular tree. Thus, organ printing is a new emerging enabling technology paradigm which represents a developmental biology-inspired alternative to classic biodegradable solid scaffold-based approaches in tissue engineering.
Keywords: Tissue engineering; Organ printing; Tissue spheroids; Tissue fusion; Bioreactor;

Can bioactivity be tested in vitro with SBF solution? by Marc Bohner; Jacques Lemaitre (2175-2179).
A large part of the scientific community has accepted the paradigm that a simulated body solution (SBF) can be used to test the bioactivity of a material. This is exemplified by the rapidly increasing number of publications using this test. The aim of this document is to demonstrate that (i) there is presently not enough scientific data to support this assumption, and (ii) even though the assumption was valid, the way the test is generally conducted leaves room for improvement. Theoretical arguments and facts supporting these statements are provided, together with possible improvements of the proposed bioactivity test.
Keywords: Bone; Bioactivity; Calcium phosphate; Bioglass; Hydroxyapatite; Simulated body fluid;

Reduction-sensitive polymers and bioconjugates for biomedical applications by Fenghua Meng; Wim E. Hennink; Zhiyuan Zhong (2180-2198).
Reduction-sensitive biodegradable polymers and conjugates have emerged as a fascinating class of biomedical materials that can be elegantly applied for intracellular triggered gene and drug delivery. The design rationale of reduction-sensitive polymers and conjugates usually involves incorporation of disulfide linkage(s) in the main chain, at the side chain, or in the cross-linker. Reduction-sensitive polymers and conjugates are characterized by an excellent stability in the circulation and in extracellular fluids, whereas they are prone to rapid degradation under a reductive environment present in intracellular compartments such as the cytoplasm and the cell nucleus. This remarkable feature renders them distinct from their hydrolytically degradable counterparts and extremely intriguing for the controlled cytoplasmic delivery of a variety of bioactive molecules including DNA, siRNA, antisense oligonucleotide (asODN), proteins, drugs, etc. This review presents recent advances in the development of reduction-sensitive biodegradable polymers and conjugates, with particular focus on the up-to-date design and chemistry of various reduction-sensitive delivery systems including liposomes, polymersomes, polymeric micelles, DNA containing nanoparticles, polyion complex micelles, nano- and micro-gels, nanotubes, and multi-layered thin films. It is evident that reduction-sensitive biodegradable polymers and conjugates are highly promising functional biomaterials that have enormous potential in formulating sophisticated drug and gene delivery systems.
Keywords: Disulfide; Reduction-sensitive; Glutathione; Biodegradation; Drug delivery; Gene transfer;

The effect of mesoporous bioactive glass on the physiochemical, biological and drug-release properties of poly(dl-lactide-co-glycolide) films by Chengtie Wu; Yogambha Ramaswamy; Yufang Zhu; Rongkun Zheng; Richard Appleyard; Andrew Howard; Hala Zreiqat (2199-2208).
Poly(lactide-co-glycolide) (PLGA) has been widely used for bone tissue regeneration. However, it lacks hydrophilicity, bioactivity and sufficient mechanical strength and its acidic degradation by-products can lead to pH decrease in the vicinity of the implants. Mesoporous bioactive glass (MBG) with highly ordered structure (pore size 2–50 nm) possesses higher bioactivity than non-mesoporous bioactive glass (BG). The aim of this study is to investigate the effect of MBG on the mechanical strength, in vitro degradation, bioactivity, cellular response and drug release of PLGA films and optimize their physicochemical, biological and drug-delivery properties for bone tissue engineering application. The surface and inner microstructure, mechanical strength and surface hydrophilicity of MBG/PLGA and BG/PLGA films were tested. Results indicated that MBG or BG was uniformly dispersed in the PLGA films. The incorporation of MBG into PLGA films significantly improved their tensile strength, modulus and surface hydrophilicity. MBG/PLGA resulted in an enhanced mechanical strength, in vitro degradation (water absorbance, weight loss and ions release), apatite-formation ability and pH stability in simulated body fluids (SBF), compared to BG/PLGA. MBG/PLGA and BG/PLGA films enhanced human osteoblastic-like cells (HOBs) attachment, spreading and proliferation compared to PLGA. HOBs differentiation was significantly upregulated when cells were cultured on 30 MBG/PLGA for 14 days, compared to 30 BG/PLGA. MBG/PLGA enhanced the accumulative release of dexamethazone (DEX) at early stages (0–200 h) compared to BG/PLGA, however, after 200 h, DEX-release rates for MBG/PLGA was slower than that of BG/PLGA. The contents of MBG in PLGA films can control the amount of DEX released. Taken together, MBG/PLGA films possessed excellent physicochemical, biological and drug-release properties, indicating their potential application for bone tissue engineering by designing 3D scaffolds according to their corresponding compositions.
Keywords: Mesoporous bioactive glass; PLGA; Osteoblasts; Dexamethazone; Drug release;

Cellular patterning on biomaterial surfaces is important in fundamental studies of cell–cell and cell–substrate interactions, and in biomedical applications such as tissue engineering, cell-based biosensors, and diagnostic devices. In this study, we combined the layer-by-layer polyelectrolyte multilayer deposition and photolithographic technique to create an easy and versatile technique for cell patterning. Poly(acrylic acid) (PAA) conjugated with 4-azidoaniline was interwoven in PAA/polyacrylamide (PAM) multilayer films. After UV irradiation through a photo mask, the UV-exposed areas were crosslinked and the unexposed areas were rinsed away by alkaline water, resulting in micropatterns. Cell patterns were formed when the cell adhesion was limited to the base substrate, but not on the multilayer films. The stability of cell patterns could be modulated by simply modification of the surface chemistry of base substrate and PEM films with conjugation of bioactive macromolecules. This technique can be also applied to other PEM systems with proper rinsing protocol, and many types of substrates. Cell co-culture systems can be also achieved by this technique.
Keywords: Mechanical compliance; Topography; Co-culture; Micropatterning;

A series of block poly(ester-urethane) poly(3/4HB-HHxHO) urethanes (abbreviated as PUHO) based on poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3/4HB-diol) and poly(3-hydroxyhexanoate-co-3-hydroxyoctanoate) (PHHxHO-diol) segments were synthesized by a facile way of melting polymerization using 1,6-hexamethylene diisocyanate (HDI) as the coupling agent, with different 3HB, 4HB, HHxHO compositions and segment lengths. The chemical structure, molecular weight and distribution were systematically characterized by 1H, 13C nuclear magnetic resonance spectrum (NMR), two-dimensional correlation spectroscopy (COSY (1H, 13C) NMR), Fourier transform infrared spectroscopy (FTIR) and gel permeation chromatography (GPC). The thermal property was studied by differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA). The hydrophilicity was investigated by static contact angle of water and CH2I2. DSC revealed that the poly(3/4HB-HHxHO) urethanes are almost amorphous with a little crystallinity (less than 6%) and T g from −23 °C to −3 °C. The polyurethanes are more hydrophobic (water contact angle 88°–117°) than the P3/4HB and PHHxHO raw materials. The lactate dehydrogenase (LDH) assay and platelet adhesion determination showed that the obtained polyurethanes have much higher platelet adhesion property than raw materials and common biodegradable polymers polylactic acid (PLA) and poly(3-hydroxybutyrate) (PHB). Hydrophobicity and crystallinity degree are important factors to affect the platelet adhesion. All the properties can be tailored by changing the composition and segment length of prepolymers P3/4HB-diol and PHHxHO-diol.
Keywords: Polyurethane; Poly(3-hydroxybutyrate-co-4-hydroxybutyrate); Melting polymerization; Characterization; Platelet adhesion;

Immunocompatibility properties of lipid–polymer hybrid nanoparticles with heterogeneous surface functional groups by Carolina Salvador-Morales; Liangfang Zhang; Robert Langer; Omid C. Farokhzad (2231-2240).
Here we report the immunological characterization of lipid–polymer hybrid nanoparticles (NPs) and propose a method to control the levels of complement activation induced by these NPs. This method consists of the highly specific modification of the NP surface with methoxyl, carboxyl, and amine groups. Hybrid NPs with methoxyl surface groups induced the lowest complement activation, whereas the NPs with amine surface groups induced the highest activation. All possible combinations among carboxyl, amine, and methoxyl groups also activated the complement system to a certain extent. All types of NPs activated the complement system primarily via the alternative pathway rather than the lectin pathway. The classical pathway was activated to a very small extent by the NPs with carboxyl and amine surface groups. Human serum and plasma protein binding studies showed that these NPs had different protein binding patterns. Studies of both complement activation and coagulation activation suggested that NPs with methoxyl surface groups might be an ideal candidate for drug delivery applications, since they are not likely to cause any immunological adverse reaction in the human body.
Keywords: Lipid–polymer hybrid nanoparticles; Surface functional groups; Complement system activation; Human plasma protein binding; Coagulation system;

Development of cell-selective films for layered co-culturing of vascular progenitor cells by Mark S.K. Chong; Jerry Chan; Mahesh Choolani; Chuen-Neng Lee; Swee-Hin Teoh (2241-2251).
Cell-sheet assemblies are currently being studied for tissue engineering. However, tissues engineered from completely biological cell sheets lack substrate cues and possess poor mechanical strength. Recent studies demonstrate the use of synthetic bioresorbable films as scaffolds that may address these issues. Here, we describe the application of a micro-thin, biaxially-stretched polycaprolactone (μXPCL) with surface modifications for layered tissue engineering, and present the results of biphasic cell-sheet constructs using surfaces optimised for specific cell types. Polyacrylic acid (PAAc) was grafted onto μXPCL film surfaces by low-pressure plasma immobilisation. This provided a surface suitable for perivascular cells, forming the medial compartment. Subsequently, endothelial progenitor cell (EPC)-selective CD34 antibody was conjugated onto the reverse surface (intimal compartment) to select and anchor EPCs for improved adhesion and proliferation. Using the blood vessel as a model, a biphasic culture system was then setup to represent a tunica intima (endothelial cells) and tunica media (smooth muscle cells). When suitable cell types were cultured in the corresponding compartments, confluent layers of the respective populations were achieved distinctively from each other. These results demonstrate the use of μXPCL films with cell-selective modifications for layered co-cultures towards the generation of stratified tissue.
Keywords: Polycaprolactone; Surface modification; Cell capture; Micro-thin films; Cell sheet engineering;

Biomimetic nanofibrous gelatin/apatite composite scaffolds for bone tissue engineering by Xiaohua Liu; Laura A. Smith; Jiang Hu; Peter X. Ma (2252-2258).
Mimicking certain features (e.g. nanoscale topography and biological cues) of natural extracellular matrix (ECM) is advantageous for the successful regeneration of damaged tissue. In this study, nanofibrous gelatin/apatite (NF-gelatin/apatite) composite scaffolds have been fabricated to mimic both the physical architecture and chemical composition of natural bone ECM. A thermally induced phase separation (TIPS) technique was developed to prepare nanofibrous gelatin (NF-gelatin) matrix. The NF-gelatin matrix mimicked natural collagen fibers and had an average fiber diameter of about 150 nm. By integrating the TIPS method with porogen leaching, three-dimensional NF-gelatin scaffolds with well-defined macropores were fabricated. In comparison to Gelfoam® (a commercial gelatin foam) with similar pore size and porosity, the NF-gelatin scaffolds exhibited a much higher surface area and mechanical strength. The surface area and compressive modulus of NF-gelatin scaffolds were more than 700 times and 10 times higher than that of Gelfoam®, respectively. The NF-gelatin scaffolds also showed excellent biocompatibility and mechanical stability. To further enhance pre-osteoblast cell differentiation as well as improving mechanical strength, bone-like apatite particles (<2 μm) were incorporated onto the surface of NF-gelatin scaffolds via a simulated body fluid (SBF) incubation process. The NF-gelatin/apatite scaffolds 5 days after SBF treatment showed significantly higher mechanical strength than NF-gelatin scaffolds 5 days after SBF treatment. Furthermore, the incorporated apatite in the NF-gelatin/apatite composite scaffold enhanced the osteogenic differentiation. The expression of BSP and OCN in the osteoblast–(NF-gelatin/apatite composite) constructs was about 5 times and 2 times higher than in the osteoblast–(NF-gelatin) constructs 4 weeks after cell culture. The biomimetic NF-gelatin/apatite scaffolds are, therefore, excellent for bone tissue engineering.
Keywords: Biomimetic; Gelatin; Nanofiber; Composite scaffold; Bone tissue engineering;

The control of anchorage-dependent cell behavior within a hydrogel/microcarrier system in an osteogenic model by Chunming Wang; Yihong Gong; Yuan Zhong; Yongchang Yao; Kai Su; Dong-An Wang (2259-2269).
The use of injectable hydrogels for tissue engineering purposes such as bone regeneration has been hampered by the mass depletion of cells after encapsulation, due to the lack of a proper interface between hydrogel matrices and osteo-progenitor cells. Efforts to graft bioactive molecules as cell attachment moieties have achieved limited success. In this study, we devised a solution to promote cellular focal adhesion within hydrogels, and elicit the mechanism behind cellular survival/death therein. We found that the fulfillment of ligation between cellular integrins and extracellular ligands, instead of the expression of integrins per se, is essential to avoid apoptosis in gel-encapsulated anchorage-dependent cells (ADCs). Absence of such ligation brought about mass cell death in our osteogenic model with osteoblasts (as representative of ADCs) and failure of osteogenic commitment of mesenchymal stem cells (as representative of anchorage-dependent progenitors). We have designed a gel-based composite system that works as a suspension of injectable cell-laden microcarriers in hydrogel, as compared to the conventional cell-suspended hydrogels. Injectable microscopic anchors (microcarriers) not only provide platforms for cellular focal adhesion but also facilitate the cells to overcome gel enlacement and fully spread out into their natural morphology. Further in vitro and in vivo osteogenic investigations show the composites to be a competent potential injectable vehicle for the conveyance of ADCs and regenerations of bone and other tissues.
Keywords: Hydrogel; Microcarrier; Anchorage-dependent cells; Apoptosis; Integrin; Osteogenesis;

Bioartificial pancreas, microencapsulation of islets of Langerhans (islets) within devices has been studied as a safe and simple technique for islet transplantation without the need for immuno-suppressive therapy. Various types of bioartificial pancreas have been proposed and developed such as microcapsule, macrocapsule and diffusion chamber types. However, these materials comprising a bioartificial pancreas are not completely inert and may induce foreign body and inflammatory reactions. The residual materials would be a problem in human body. Here we propose an alternative method for microencapsulation of islets with a layer of living cells. We immobilized HEK293 cells (human endoderm kidney cell line) to the islet surface using amphiphilic poly(ethylene glycol)-conjugated phospholid derivative and biotin/streptavidin reaction and encapsulated islets with a cell layer by culture. No necrosis of islet cells at the center was seen after microencapsulation with a layer of living cells. Insulin secretion ability by glucose stimulation was well maintained on these cell-encapsulated islets.
Keywords: Bioartificial pancreas; Islet; Cell-encapsulation; Microencapsulation; Poly(ethylene glycol)–lipid (PEG–lipid);

The effect of a layer-by-layer chitosan–heparin coating on the endothelialization and coagulation properties of a coronary stent system by Sheng Meng; Zongjun Liu; Li Shen; Zhang Guo; Laisheng L. Chou; Wei Zhong; Qiangguo Du; Junbo Ge (2276-2283).
A biomacromolecular layer-by-layer coating process of chitosan/heparin onto a coronary stent is designed for the acceleration of the re-endothelialization and healing process after coronary stent deployment. The results of in vitro culturing of porcine iliac artery endothelial cells as well as the measurements of the APTT, PT and TT supported the rationale that the combination of chitosan and heparin could bring both endothelial cell compatibility and haemocompatibility to the stent surface. A porcine coronary injury model and arteriovenous shunt model were used for the further evaluation of the application of this kind of surface-modified stainless steel stent in vivo. The final results proved that this facile coating approach could significantly promote re-endothelialization and was safer compared with bare metal stents for its much improved anticoagulation property.
Keywords: Stent; Haemocompatibility; Endothelialization; Self assembly;

Bienzyme functionalized three-layer composite magnetic nanoparticles for electrochemical immunosensors by Ying Zhuo; Pei-Xi Yuan; Ruo Yuan; Ya-Qin Chai; Cheng-Lin Hong (2284-2290).
The preparation, characterization and application of a three-layer magnetic nanoparticle composed of an Fe3O4 magnetic core, a Prussian Blue (PB) interlayer and a gold shell (it can be abbreviated as Au–PB–Fe3O4) for an ultrasensitive and reproducible electrochemical immunosensing fabrication were described for the first time in this work. With the employment of the Au–PB–Fe3O4 nanoparticle, a new signal amplification strategy was developed based on bienzyme (horseradish peroxidase and glucose oxidase) functionalized Au–PB–Fe3O4 nanoparticles for an electrochemical immunosensing fabrication by using Carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) as model systems, respectively. The experiment results show that the multilabeled Au–PB–Fe3O4 nanoparticles exhibit satisfying redox electrochemical activity and high enzyme catalysis activity, which predetermine their utility in high sensitivity antibody detection schemes. Furthermore, this immunosensor could be regenerated by simply using an external magnetic field which ensured a reproducible immunosensor with high sensitivity.
Keywords: Magnetic nanocomposite; Biosensor; Enzyme; Electrochemistry;

The effect of microstructured surfaces and laminin-derived peptide coatings on soft tissue interactions with titanium dental implants by Sandra Werner; Olivier Huck; Benoît Frisch; Dominique Vautier; René Elkaim; Jean-Claude Voegel; Gérard Brunel; Henri Tenenbaum (2291-2301).
In the present study, we investigated the dental implant protection from peri-implant inflammation by improving the soft tissue adhesion on the titanium surface. Porous titanium was used to create, at the level of the transmucosal part of the implants (the “neck”), a microstructured 3-dimensional surface that would tightly seal the interface between the implant and soft tissue. Cell-specific adhesion properties were induced via an adhesion peptide derived from laminin-5 coupled to native or cross-linked PLL/PGA multilayered polyelectrolyte films (MPFs), which are used for biomedical device coatings. Porous titanium exhibited good cell-adhesion properties, but the colonisation of the material was further improved by a coating with laminin-5 functionalised MPFs and especially with (PLL/PGA)6,5–PGA–peptide film. Focal contact formation was observed on cross-linked architectures, reflecting cell anchorage on these surfaces. In contrast, when seeded on laminin-5-functionalised native films, epithelial cells formed only very diffuse focal contacts, but adhered via hemidesmosome formation. In vivo experiments confirmed that the porous titanium was colonised by cells of soft tissue. Altogether, the results indicate that the microstructure of the implant neck combined with a specific bioactive coating could constitute efficient routes to improve the integration of soft tissue on titanium dental implants, which could significantly protect implants from peri-implant inflammation and enhance long-term implant stabilisation.
Keywords: Adhesion molecule; Dental implant; Epithelial cells; Laminin-5; Microstructured titanium; Polyelectrolyte films;

Convection-enhanced delivery of nanocarriers for the treatment of brain tumors by Emilie Allard; Catherine Passirani; Jean-Pierre Benoit (2302-2318).
Primary brain tumors have a significant infiltrative capacity as their reappearance after resection usually occurs within 2 cm of the tumor margin. Local delivery method such as Convection-Enhanced Delivery (CED) has been introduced to avoid this recurrence by delivering active molecules via positive-pressure methods. For an efficient infusion, the distribution volume of the drug has to be optimized while avoiding backflow, since this is responsible for side effects and a reduction of therapeutic efficacy. The encapsulation of the drug infused in nanosized structures can be considered, which would lead to a reduction of both toxicity of the treatment and infusion time during CED. In the present review, we will firstly discuss the technical approach of CED with regard to catheter design and brain characteristics; secondly, we will describe the ‘ideal’ nanocarrier in terms of size, surface properties, and interaction with the extracellular matrix for optimal diffusion in the brain parenchyma. We also discuss preclinical and clinical applications of this new method.
Keywords: Brain tumor; Convection-enhanced delivery; Backflow; Distribution volume; Nanocarrier; Anticancer treatment;

The protection of cells from nitric oxide-mediated apoptotic death by mechanochemically synthesized fullerene (C60) nanoparticles by Maja S. Misirkic; Biljana M. Todorovic-Markovic; Ljubica M. Vucicevic; Kristina D. Janjetovic; Vukoman R. Jokanovic; Miroslav D. Dramicanin; Zoran M. Markovic; Vladimir S. Trajkovic (2319-2328).
The influence of fullerene (C60) nanoparticles on the cytotoxicity of a highly reactive free radical nitric oxide (NO) was investigated. Fullerene nanoparticles were prepared by mechanochemically assisted complexation with anionic surfactant sodium dodecyl sulfate, macrocyclic oligosaccharide γ-cyclodextrin or the copolymer ethylene vinyl acetate–ethylene vinyl versatate. C60 nanoparticles were characterized by UV–vis and atomic force microscopy. While readily internalized by mouse L929 fibroblasts, C60 nanoparticles were not cytotoxic. Moreover, they partially protected L929 cells from the cytotoxic effect of NO-releasing compounds sodium nitroprusside (SNP), S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO) and 3-morpholino-sydnonimine (SIN-1). C60 nanoparticles reduced SNP-induced apoptotic cell death by preventing mitochondrial depolarization, caspase activation, cell membrane phosphatidylserine exposure and DNA fragmentation. The protective action of C60 nanoparticles was not exerted via direct interaction with NO, but through neutralization of mitochondria-produced superoxide radical in NO-treated cells, as demonstrated by using different redox-sensitive reporter fluorochromes. These data suggest that C60 complexes with appropriate host molecules might be plausible candidates for preventing NO-mediated cell injury in inflammatory/autoimmune disorders.
Keywords: Carbon; Nanoparticles; Nitric oxide; Cytotoxicity; Apoptosis;

In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery by Kiran Sonaje; Yu-Hsin Lin; Jyuhn-Huarng Juang; Shiaw-Pyng Wey; Chiung-Tong Chen; Hsing-Wen Sung (2329-2339).
A variety of approaches have been studied in the past to overcome the problems encountered with the oral delivery of insulin, but with little success. In this study, self-assembled nanoparticles (NPs) with a pH-sensitive characteristic were prepared by mixing the anionic poly-γ-glutamic acid solution with the cationic chitosan solution in the presence of MgSO4 and sodium tripolyphosphate. The in vitro results found that the transport of insulin across Caco-2 cell monolayers by NPs appeared to be pH-dependent; with increasing pH, the amount of insulin transported decreased significantly. An in vivo toxicity study was performed to establish the safety of the prepared NPs after oral administration. Additionally, the impact of orally administered NPs on the pharmacodynamics (PD) and pharmacokinetics (PK) of insulin was evaluated in a diabetic rat model. The in vivo results indicated that the prepared NPs could effectively adhere on the mucosal surface and their constituted components were able to infiltrate into the mucosal cell membrane. The toxicity study indicated that the NPs were well tolerated even at a dose 18 times higher than that used in the PD/PK study. Oral administration of insulin-loaded NPs demonstrated a significant hypoglycemic action for at least 10 h in diabetic rats and the corresponding relative bioavailability of insulin was found to be 15.1 ± 0.9%. These findings suggest that the NPs prepared in the study are a promising vehicle for oral delivery of insulin.
Keywords: Nanoparticle; Toxicity; Efficacy; Insulin; Tight junction;

Long-circulating polymeric nanoparticles bearing a combinatorial coating of PEG and water-soluble chitosan by Yan Sheng; Changsheng Liu; Yuan Yuan; Xinyi Tao; Fan Yang; Xiaoqian Shan; Huanjun Zhou; Feng Xu (2340-2348).
A major obstacle in the development of polymeric nanoparticles (NPs) as effective drug delivery vesicles is the rapid clearance from blood. In order to realize a significant prolongation in blood circulation, a combinatorial design, covalent attachment of polyethylene glycol (PEG) to polylactic acid (PLA) and physical adsorption of water-soluble chitosan (WSC) to particle surface, has been developed for surface modification of PLA NPs. Two types of WSC, cationic partially deacetylated chitin (PDC) and anionic N-carboxy propionyl chitosan sodium (CPCTS) were investigated. All the NPs formulated in the size range of 100–200 nm were prepared by a modified w/o/w technique and physicochemically characterized. In vitro phagocytosis by mouse peritoneal macrophage (MPM), in vivo blood clearance and biodistribution following intravenous administration in mice, of these NPs labeled with 6-coumarin, were evaluated. The presence of WSC, whether alone or with PEG, highly improved the surface hydrophilicity as well as suspension stability of NPs. Their surface charge was greatly affected by the WSC coating, being close to neutrality for PEG/PDC NPs and highly negative in the case of PEG/CPCTS NPs. In comparison to NPs treated with PEG or WSC alone, the synergistic action of PEG and WSC strongly inhibited the macrophage uptake and extended the circulation half-life (t 1/2) with concomitant reduced liver sequestration. Particularly, PEG/PDC NPs showed the most striking result with regard to their performance in vitro and in vivo. Calculated t 1/2 of PEG/PDC NPs and PEG/CPCTS NPs was 63.5 h and 7.1 h, respectively, much longer than that of control PEG/PVA NPs (1.1 h). More WSC materials need to be evaluated, but the present data suggest that, a combinatorial coating of PEG and PDC greatly prolongs the systemic circulation of NPs and represents a significant step in the development of long-circulating drug delivery carriers.
Keywords: Polymeric nanoparticles; Polyethylene glycol; Water-soluble chitosan; Long-circulating; Biodistribution;

Thermosensitive poly(organophosphazene)–paclitaxel conjugate gels for antitumor applications by ChangJu Chun; Sun Mi Lee; Sang Yoon Kim; Han Kwang Yang; Soo-Chang Song (2349-2360).
A poly(organophosphazene)–PTX conjugate was synthesized by a covalent ester linkage between PTX and carboxylic acid-terminated poly(organophosphazene), which can be readily modified by various hydrophobic, hydrophilic, and other functional substitutes. The physicochemical properties, hydrolytic degradation and PTX release behaviors of the polymer–PTX conjugate were characterized, in addition to the in vitro and in vivo antitumor activities. The aqueous solutions of these conjugates showed a sol–gel transition behavior that depended on temperature changes. The in vitro antitumor activity of the polymer–PTX conjugate was investigated by an MTT assay against human tumor cell lines. From the in vivo antitumor activity studies with tumor-induced (xenografted) nude mice, the polymer–paclitaxel conjugate hydrogels after local injection at the tumor site were shown to inhibit tumor growth more effectively and longer than paclitaxel and saline alone, indicating that the tumor-active paclitaxel from the polymer–PTX conjugate hydrogel is released slowly over a longer period of time and effectively accumulated locally in the tumor sites. These combined observations suggest that this poly(organophosphazene)–PTX conjugate holds promise for use in clinical studies as single and/or combination therapies.
Keywords: Biodegradable; Thermosensitive; Hydrogel; Polymer–drug conjugate; Paclitaxel; Antitumor activity;

Tissue engineering scaffolds with complex geometries can provide an architecture that directs tissue formation. Drug delivery from these scaffolds to promote regeneration is often challenging due to the complex fabrication processes. Surface-mediated DNA delivery from multiple channel bridges was applied to deliver lipoplexes in vivo to the injured spinal cord. The surface properties of the polymer, DNA deposition with or without drying, and the presence of ECM components were investigated. In vitro studies revealed that fibronectin produced greater expression levels and immobilization efficiencies compared with collagen, laminin, and no coating. In addition, lipoplex incubation on ECM-coated PLG increased expression relative to either of the drying methods. Additionally, the incubation method had more homogeneously distributed lipoplexes and a higher number of transfected cells relative to the dried conditions. Translation to three-dimensional bridges led to high levels of transgene expression in vitro. In vivo, lipoplexes immobilized to the bridge produced transgene expression levels in a rat spinal cord hemisection model that were 2-fold greater than naked plasmid. Additionally, expression with lipoplexes persisted for at least three weeks. Surface-mediated delivery can be applied to scaffolds with complex geometries to promote transgene expression in vivo.
Keywords: Multiple channel bridge; Gene therapy; Spinal cord regeneration; ECM; Lipoplexes; Non-viral vector;

Cationic glycolipids with cyclic and open galactose head groups for the selective targeting of genes to mouse liver by Rajesh Mukthavaram; Srujan Marepally; Mahidhar Y. Venkata; Gangamodi N. Vegi; Ramakrishna Sistla; Arabinda Chaudhuri (2369-2384).
Toward probing an hitherto unexplored structure–activity issue namely, the relative in vitro and in vivo efficacies of cationic glycolipids with cyclic and acyclic sugar heads for targeting of genes to liver, we have designed and synthesized two novel series of cationic glycolipids with cyclic (lipids 15) and open d-galactose heads (lipids 610) containing varying spacer arm lengths in between the sugar and positively charged nitrogen atoms. Among the cyclic glycolipids, lipid 3 with six methylene units spacer in between the quaternary nitrogen atom and among the glycolipids with the open-sugar heads, lipid 6 with only two methylene units spacer were found to be the most efficacious in targeting genes to cultured HepG2 (human hepatocarcinoma cells) and primary hepatocytes. Findings in the fluorescence resonance energy transfer (FRET) studies revealed biomembrane fusibilities as important physico-chemical parameters behind the varying spacer arm dependencies in the two series. Importantly, both the serum compatible glycolipids 3 &6 were found to be equally efficacious in selectively targeting genes to mouse livers under systemic settings. The significantly reduced efficiencies of the glycolipids 3 &6 in transfecting primary hepatocytes as well as mice pretreated with asialofetuin (the ligands of asialoglycoprotein receptors) support the notion that the cellular uptake of the lipoplexes prepared from both the open and the cyclic sugar-head series is mediated via asialoglycoprotein receptor. In summary, our present findings demonstrate for the first time that cationic glycolipids with cyclic sugar-head require longer spacer arms than their acyclic sugar-head counterparts for efficient gene transfection and both the series hold equal promise for selective gene targeting to liver under systemic settings.
Keywords: Liver; Hepatocytes; Liposomes; Gene transfer; Selective liver transfection; Asialoglycoprotein receptor;

The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects by Stephan Vogt; Gabriele Wexel; Thomas Tischer; Ulrike Schillinger; Peter Ueblacker; Bettina Wagner; Daniel Hensler; Jonas Wilisch; Christopher Geis; Daniela Wübbenhorst; Joachim Aigner; Michael Gerg; Achim Krüger; Gian M. Salzmann; Vladimir Martinek; Martina Anton; Christian Plank; Andreas B. Imhoff; Bernd Gansbacher (2385-2392).
Growth factors like BMP2 have been tested for osteochondral repair, but transfer methods used until now were insufficient. Therefore, the aim of this study was to analyse if stable BMP2 expression after retroviral vector (Bullet) transduction is able to regenerate osteochondral defects in rabbits. Fibrin clots colonized by control or BMP2-transduced chondrocytes were generated for in vitro experiments and implantation into standardized corresponding osteochondral defects (n  = 32) in the rabbit trochlea. After 4 and 12 weeks repair tissue was analysed by histology (HE, alcian-blue, toluidine-blue), immunohistochemistry (Col1, Col2, aggrecan, aggrecan-link protein), ELISA (BMP2), and quantitative RT-PCR (BMP2, Col1, Col2, Col10, Cbfa1, Sox9). In vitro clots were also analysed by BMP2-ELISA, histology (alcian-blue), quantitative RT-PCR and in addition by electron microscopy. BMP2 increased Col2 expression, proteoglycan production and cell size in vitro. BMP2 transduction by Bullet was efficient and gene expression was stable in vivo over at least 12 weeks. Proteoglycan content and ICRS-score of repair tissue were improved by BMP2 after 4 and 12 weeks and Col2 expression after 4 weeks compared to controls. However, in spite of stable BMP2 expression, a complete repair of osteochondral defects could not be demonstrated. Therefore, BMP2 is not suitable to regenerate osteochondral lesions completely.
Keywords: Retroviral vector; Fibrin clot; BMP2; Osteochondral defect; Hyaline; Cartilage;

The influence of extracellular matrix derived from skeletal muscle tissue on the proliferation and differentiation of myogenic progenitor cells ex vivo by Matthew M. Stern; Regina L. Myers; Nevin Hammam; Kathryn A. Stern; Daniel Eberli; Stephen B. Kritchevsky; Shay Soker; Mark Van Dyke (2393-2399).
Skeletal muscle relies upon regeneration to maintain homeostasis and repair injury. This process involves the recruitment of the tissue's resident stem cell, the muscle progenitor cell, and a subsequent proliferative response by newly generated myoblasts, which must then align and fuse to generate new muscle fibers. During regeneration, cells rely on environmental input for direction. Extracellular matrix (ECM) represents a crucial component of a cell's microenvironment that aids in guiding muscle regeneration. We hypothesized that ECM extracted from skeletal muscle would provide muscle progenitor cells and myoblasts with an ideal substrate for growth and differentiation ex vivo. To test this hypothesis, we developed a method to extract ECM from the large thigh muscles of adult rats and present it to cells as a surface coating. Myogenic cells cultured on ECM extract experienced enhanced proliferation and differentiation relative to standard growth surfaces. As the methodology can be applied to any size muscle, these results demonstrate that bioactive ECM can be readily obtained from skeletal muscle and used to develop biomaterials that enhance muscle regeneration. Furthermore, the model system demonstrated here can be applied to the study of interactions between the ECM of a particular tissue and a cell population of interest.
Keywords: Extracellular matrix; Muscle; Progenitor cell; Cell culture; Cell proliferation;

Co-assembling peptides as defined matrices for endothelial cells by Jangwook P. Jung; Arun K. Nagaraj; Emily K. Fox; Jai S. Rudra; Jason M. Devgun; Joel H. Collier (2400-2410).
Self-assembling peptides and peptide derivatives bearing cell-binding ligands are increasingly being investigated as defined cell culture matrices and as scaffolds for regenerative medicine. In order to systematically refine such scaffolds to elicit specific desired cell behaviors, ligand display should ideally be achieved without inadvertently altering other physicochemical properties such as viscoelasticity. Moreover, for in vivo applications, self-assembled biomaterials must exhibit low immunogenicity. In the present study, multi-peptide co-assembling hydrogels based on the β-sheet fibrillizing peptide Q11 (QQKFQFQFEQQ) were designed such that they presented RGDS or IKVAV ligands on their fibril surfaces. In co-assemblies of the ligand-bearing peptides with Q11, ligand incorporation levels capable of influencing the attachment, spreading, morphology, and growth of human umbilical vein endothelial cells (HUVECs) did not significantly alter the materials' fibrillization, β-turn secondary structure, or stiffness. RGDS-Q11 specifically increased HUVEC attachment, spreading, and growth when co-assembled into Q11 gels, whereas IKVAV-Q11 exerted a more subtle influence on attachment and morphology. Additionally, Q11 and RGDS-Q11 were minimally immunogenic in mice, making Q11-based biomaterials attractive candidates for further investigation as defined, modular extracellular matrices for applications in vitro and in vivo.
Keywords: Self-assembly; Biomimetic material; Biomaterial immunogenicity; Regenerative medicine; 3-D culture;

Micromechanics of bone tissue-engineering scaffolds, based on resolution error-cleared computer tomography by Stefan Scheiner; Raffaele Sinibaldi; Bernhard Pichler; Vladimir Komlev; Chiara Renghini; Chiara Vitale-Brovarone; Franco Rustichelli; Christian Hellmich (2411-2419).
Synchrotron radiation micro-computed tomography (SRμCT) revealed the microstructure of a CEL2 glass–ceramic scaffold with macropores of several hundred microns characteristic length, in terms of the voxel-by-voxel 3D distribution of the attenuation coefficients throughout the scanned space. The probability density function of all attenuation coefficients related to the macroporous space inside the scaffold gives access to the tomograph-specific machine error included in the SRμCT measurements (also referred to as instrumental resolution function). After Lorentz function-based clearing of the measured CT data from the systematic resolution error, the voxel-specific attenuation information of the voxels representing the solid skeleton is translated into the composition of the material inside one voxel, in terms of the nanoporosity embedded in a dense CEL2 glass–ceramic matrix. Based on voxel-invariant elastic properties of dense CEL2 glass–ceramic, continuum micromechanics allows for translation of the voxel-specific nanoporosity into voxel-specific elastic properties. They serve as input for Finite Element analyses of the scaffold structure. Young's modulus of a specific CT-scanned macroporous scaffold sample, predicted from a Finite Element simulation of a uniaxial compression test, agrees well with the experimental value obtained from an ultrasonic test on the same sample. This highlights the satisfactory predictive capabilities of the presented approach.
Keywords: CEL2 glass–ceramic; Scaffold; Bone tissue engineering; Continuum micromechanics; Microtomography; Finite element method;

SnapShot (2421-2423).