Biomaterials (v.29, #16)

The role of oxygen termination of nanocrystalline diamond on immobilisation of BMP-2 and subsequent bone formation by Frank R. Kloss; Robert Gassner; Johannes Preiner; Andreas Ebner; Karin Larsson; Oliver Hächl; Tarkan Tuli; Michael Rasse; Doris Moser; Klaus Laimer; Eike A. Nickel; Gerhard Laschober; Regina Brunauer; Günther Klima; Peter Hinterdorfer; Doris Steinmüller-Nethl; Günter Lepperdinger (2433-2442).
Medical implants are increasingly often inserted into bone of frail patients, who are advanced in years. Due to age, severe trauma or pathology-related bone changes, osseous healing at the implant site is frequently limited. We were able to demonstrate that coating of endosseous implants with nanocrystalline diamond (NCD) allows stable functionalization by means of physisorption with BMP-2. Strong physisorption was shown to be directly related to the unique properties of NCD, and BMP-2 in its active form interacted strongly when NCD was oxygen-terminated. The binding of the protein was monitored under physiological conditions by single molecule force spectroscopy, and the respective adsorption energies were further substantiated by force-field-calculations. Implant surfaces refined in such a manner yielded enhanced osseointegration in vivo, when inserted into sheep calvaria. Our results further suggest that this technical advancement can be readily applied in clinical therapies with regard to bone healing, since primary human mesenchymal stromal cells strongly activated the expression of osteogenic markers when being cultivated on NCD physisorbed with physiological amounts of BMP-2.
Keywords: Nanocrystalline diamond (NCD); Physisorption; Bone morphogenetic protein-2 (BMP-2); Bone regeneration; Mesenchymal stem cells; Nanotopography;

The preparation and characterization of a commercial biomedical-grade polyurethane (Tecophilic®, SP-93A-100) material possessing covalently linked copper(II)–cyclen moieties as a nitric oxide (NO) generating polymer are described. Chemiluminescence NO measurements demonstrate that the prepared polymer can decompose endogenous S-nitrosothiols (RSNOs) such as S-nitrosoglutathione and S-nitrosocysteine to NO in the presence of thiol reducing agents (RSHs; e.g., glutathione and cysteine) at physiological pH. Since such RSNO and RSH species already exist in blood, the proposed polymer is capable of spontaneously generating NO when in contact with fresh blood. This is demonstrated by utilizing the polymer as an outer coating at the distal end of an amperometric NO sensor to create a device that generates response toward the RSNO species in the blood. This polymer possesses the combined benefits of a commercial biomedical-grade polyurethane with the ability to generate biologically active NO when in contact with blood, and thus may serve as a useful coating to improve the hemocompatibility of various medical devices.
Keywords: Copper; Hemocompatibility; Nitric oxide; Polyurethane; S-nitrosothiols;

The thrombogenicity of human umbilical vein endothelial cell seeded collagen modules by Alison P. McGuigan; Michael V. Sefton (2453-2463).
Modular tissue-engineered constructs are assembled from sub-mm sized cylindrical collagen gel modules which are covered with a surface layer of human umbilical vein endothelial cells (HUVEC). The resulting construct is permeated by a network of interconnected endothelial cell lined channels to facilitate blood perfusion and nutrient delivery. This design strategy relies critically on the endothelial cells' layer behaving in a non-thrombogenic manner on the module surface and the objective here was to characterize this thrombogenicity. HUVEC prolonged clotting times in whole blood-module mixtures, and enabled slightly heparinized whole blood perfusion of an assembled modular construct in vitro with no increase in platelet loss compared to background levels. Flow cytometry and scanning electron microscopy indicated that HUVEC seeded modules reduced platelet activation and deposition but not leukocyte activation, compared to collagen only modules. Plasma recalcification times on non-stimulated HUVEC were longer compared to stimulated HUVEC but not different than that on collagen only module films and were not prolonged by incubation with a tissue factor blocking antibody. Together these data suggest that a functional non-thrombogenic layer of EC was generated on the module surface and that this layer should be sufficient to maintain continuous blood flow through an engineered modular tissue. In/ex vivo studies are warranted to confirm this conclusion.
Keywords: Thrombogenicity; Tissue engineering; Endothelial cells; Whole blood; Collagen; Vascularization;

Introduction of enzymatically degradable poly(trimethylene carbonate) microspheres into an injectable calcium phosphate cement by Wouter J.E.M. Habraken; Zheng Zhang; Joop G.C. Wolke; Dirk W. Grijpma; Antonios G. Mikos; Jan Feijen; John A. Jansen (2464-2476).
Poly(trimethylene carbonate) (PTMC) is an enzymatically degradable polyester with rubber-like properties. Introduction of this polymer into an injectable calcium phosphate bone cement can therefore be used to introduce macroporosity into the cement for tissue engineering purposes as well as to improve mechanical properties. Aim of this study was to investigate calcium phosphate cements with incorporated PTMC microspheres (PTMC CPCs) on their physical/mechanical properties and in vitro degradation characteristics. Therefore, composites were tested on setting time and mechanical strength as well as subjected to phosphate buffered saline (PBS) and enzyme containing medium. PTMC CPCs (12.5 and 25 wt%) with molecular weights of 52.7 kg mol−1 and 176.2 kg mol−1 were prepared, which showed initial setting times similar to that of original CPC. Though compression strength decreased upon incorporation of PTMC microspheres, elastic properties were improved as strain-at-yield increased with increasing content of microspheres. Sustained degradation of the microspheres inside PTMC CPC occurred when incubated in the enzymatic environment, but not in PBS, which resulted in an interconnected macroporosity for the 25 wt% composites.
Keywords: Calcium phosphate cement; Poly(trimethylene carbonate); Microspheres; Degradation; Mechanical strength;

Substrate architecture and fluid-induced shear stress during chondrocyte seeding: Role of α5β1 integrin by Caroline G. Spiteri; Edmond W.K. Young; Craig A. Simmons; Rita A. Kandel; Robert M. Pilliar (2477-2489).
Chondrocyte behaviour has been shown previously to be influenced by the architecture of the substrate on which the cells are grown. Chondrocytes cultured on fully porous titanium alloy substrates showed greater spreading and more matrix accumulation when compared to cells grown on porous-coated substrates with solid bases. We hypothesized that these features developed because of differences in fluid-induced shear stresses due to substrate architecture and that integrins mediate these responses. Computational fluid dynamics analyses predicted that cells on fully porous substrates experience time-dependent shear stresses that differ from those experienced by cells on porous-coated substrates with solid bases where media flow-through is restricted. To validate this model, the seeding protocol was modulated to affect fluid flow and this affected cell spreading and matrix accumulation as predicted. Integrin blocking experiments revealed that α5β1 integrins regulated cell shape under these two conditions and when cell spreading was prevented the increased accumulation of collagen and proteoglycans by chondrocytes seeded on fully porous substrates did not occur. Identifying the substrate-induced mechanical and molecular mechanisms that influence chondrocyte behaviour and tissue formation may ultimately lead to the formation of a tissue that more closely resembles natural articular cartilage.
Keywords: Cartilage tissue engineering; Cell spreading; Computational fluid dynamics; Integrin; Titanium alloy;

The use of chondrogenic differentiation drugs to induce stem cell differentiation using double bead microsphere structure by Kyeongsoon Park; Ji Sun Park; Dae Gyun Woo; Han Na Yang; Hyung-Min Chung; Keun-Hong Park (2490-2500).
A double bead microsphere device that functions as a 3D scaffold and also delivers bioactive molecules to cells has been developed. The system we developed consists of large polymeric microspheres that were loaded with dexamethasone (DEXA) and coated with DHEA, which were physically immobilized using a layer-by-layer (LbL) system. The initial step in this strategy involves the creation of microparticles that contain DHEA, which were simply produced using a water-in-oil-in-water (W/O/W) emulsion method. In the second step, small sized microparticles containing DHEA were coated onto positively-charged poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres that contained DEXA pretreated with poly(ethyleneimmine) (PEI). Microsphere constructs that contain DEXA and DHEA showed a significantly higher number of specific lacuna phenotypes at the end of a 4-week study in vitro and at the end of a 6-week study in vivo, irrespective of the presence of DEXA and DHEA. Therefore, the dual delivery of DEXA and DHEA can be used to engineer inflammation-free tissue in the vicinity of the implant. These double beaded PLGA microsphere constructs containing DEXA and DHEA show promise as coatings for implantable biomedical devices to improve biocompatibility and ensure in vivo performance.
Keywords: Microsphere; Scaffold; LbL; Cell differentiation; DEXA; DHEA;

Many glandular organs are developed by branching morphogenesis, an efficient and ubiquitous process for creating a larger cellular area for metabolic requirement. To regenerate a glandular organ, such as salivary glands, recapitulation of branching processes may be requisite. At present, the roles of biomaterials in regenerative branching have never been thoroughly explored. By culturing the embryonal submandibular gland (SMG) on different substrata, including polyvinyl alcohol (PVA), chitosan, and polycarbonate (PC), this study demonstrated for the first time that chitosan was capable of providing a more preferential environment for salivary gland branch formation. After culturing SMG explants on chitosan membranes, secreted extracellular matrices distributed in a reticular manner and formed thicker fibers beyond the extents of cell attachment, which were not found in PVA and PC. In the subsequent culture of explants just on the used chitosan substrates, these conditioned membranes were able to further enhance SMG branching. The fact that the promoting effects were eliminated with collagenase treatment and type I and type III collagen were identified within the adherent fibrillar extracellular matrix raised the possibility that the stimulating factors were collagen-originated. This indicates that, for SMG, chitosan is a bioactive substratum which enables cells to synthesize and deposit essential extracellular matrix, paving an important way for ensuing branch formation. Accordingly, the current study provides a larger scope to use chitosan as a biomaterial for recapitulating branching, which might be useful for the scaffold design of salivary gland regeneration.
Keywords: Salivary glands; Submandibular gland (SMG); Biomaterials; Chitosan; Collagen; Branching morphogenesis;

Long-term bone tissue reaction to polyethylene oxide/polybutylene terephthalate copolymer (Polyactive®) in metacarpophalangeal joint reconstruction by Eero Waris; Nureddin Ashammakhi; Mauri Lehtimäki; Riitta-Mari Tulamo; Pertti Törmälä; Minna Kellomäki; Yrjö T. Konttinen (2509-2515).
The poly-l/d-lactide 96/4 joint scaffolds are used to engineer fibrous tissue joints in situ for the reconstruction of metacarpophalangeal joints. In this experimental study, a supplementary elastomeric stem made of Polyactive® 1000PEO70PBT30 (a segmented block copolymer of polyethylene oxide and polybutylene terephtalate with 70/30 PEO/PBT ratio) was used to anchor the joint scaffold in the arthroplasty space. Eleven resected fifth metacarpophalangeal joints of minipig were reconstructed and evaluated radiologically and histologically for 3 years. Plain joint scaffold and Swanson silicone implant arthroplasties (11 of each) in metacarpophalangeal joints of minipig served as controls. Altogether fore limbs of eighteen minipigs were operated for the study. Deleterious tissue reaction with dramatic signs of osteolysis and inflammatory foreign-body reaction was observed around the Polyactive® stems. The mean maximum diameter of the osteolytic stem cavity was statistically wider when compared to the mean maximum diameter of Swanson implant group during the first postoperative year. Numerous osteoclasts were found at the margins of the osteolytic areas. No direct bone contact could be seen. At 1 year osteoblastic regeneration and formation of new trabecular bone followed. Finally the foreign-body reaction settled, but the adjoining bones were at this stage highly sclerotic and composed of coarse trabeculae. In contrary to previous in vivo studies suggesting biocompatibility, osteoconductivity and capability to bond to bone, Polyactive® 1000PEO70PBT30 stem in this setting caused massive osteolytic lesions and foreign-body reactions.
Keywords: Biodegradation; Bone; Foreign-body response; Osteolysis;

A surface-engineered particulate delivery system for exogenous antigens was developed in this study. Poly(d,l-lactide-co-glycolide) (PLGA) microparticles containing ovalbumin (OVA) or fluorescein isothiocyanate-conjugated bovine serum albumin (FITC–BSA) were fabricated by the double emulsion and solvent evaporation method. Encapsulation of the PLGA microparticles was performed by physisorption of multilayers of oppositely charged polyelectrolytes, including polyethylenimine (PEI) and dextran sulfate. Surface charges of the particles after layer-by-layer (LbL) adsorption were determined by the zeta potential measurements. The uptake of these particles by the J774A.1 murine macrophages was examined by fluorescence microscopy. Generation of reactive oxygen species (ROS) in J774A.1 cells was determined by flow cytometry using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) and hydroethidine (HE). Antigen presentation assays were performed in B3Z cells, a hybridoma of OVA-specific CD8+ T cells. Results obtained in this study demonstrated an effective ingestion of the PLGA microparticles and enhanced production of ROS in J774A.1 murine macrophages. Treatment of murine bone marrow-derived dendritic cells (BMDCs) with polyelectrolyte-encapsulated PLGA microparticles resulted in an in vitro activation of B3Z cells, demonstrating the feasibility of induction of adaptive immunity for class I major histocompatibility complex (MHC) by surface engineering of microparticulate vaccine delivery.
Keywords: Antigen presentation; Layer-by-layer; PLGA microparticles; Polyelectrolytes; Encapsulation;

Functionalized injectable hydrogels for controlled insulin delivery by Dai P. Huynh; Minh K. Nguyen; Bong S. Pi; Min S. Kim; Su Y. Chae; Kang C. Lee; Bong S. Kim; Sung W. Kim; Doo S. Lee (2527-2534).
The concept of this research is using poly(β-amino ester) (PAE) as a duo-functional group for synthesis of the novel sensitive injectable hydrogel for controlled drug/protein delivery. Firstly, PAE made of 1,4-butanediol diacrylate and 4,4′-trimethylene dipiperidine is used as a pH-sensitive moiety to conjugat to the temperature-sensitive biodegradable triblock copolymer of poly(ethylene glycol)–poly(ɛ-caprolactone) (PCL–PEG–PCL) to manufacture pH/temperature-sensitive injectable hydrogel of pentablock copolymer PAE–PCL–PEG–PCL–PAE. Furthermore, the cationic nature of PAE is used as the second function to make the ionic complexes with anionic biomolecule loaded into the hydrogel such as insulin. As a result, the release of drug/protein from this hydrogel device can be controlled by the degradation of copolymer. Sol–gel phase transition behavior of PAE–PCL–PEG–PCL–PAE block copolymer was investigated; the results showed that the aqueous media of the pentablock copolymer changed from a sol to a gel phase with increasing temperature and pH. The effect of anionic biomolecule such as insulin on sol–gel phase transition, degradation of the complex gel of the material with insulin was studied in vitro. Then the schematic of the ionic complexes between positive charges in PAE and the negatively charges in protein was simulated. In addition, the mechanism of controlled release behavior of insulin from the complex gel was supposed, which includes the chemically-controlled and diffusion-controlled stages. To prove the simulations, the cumulative release of the protein from the complex gel was investigated in vitro with different methods. Furthermore, the pharmacokinetic release of insulin from the complex gel in vivo on male Sprague-Dawley (SD) rats was compared with that from triblock copolymer hydrogel of PCL–PEG–PCL.
Keywords: pH/temperature-sensitive; Hydrogel; Insulin delivery; Controlled release;

Poly(β-amino ester) as a carrier for si/shRNA delivery in lung cancer cells by Dhananjay Jere; Cheng-Xiong Xu; Rohidas Arote; Cheol-Heui Yun; Myung-Haing Cho; Chong-Su Cho (2535-2547).
Efficient delivery of small interfering RNA (siRNA) or small hairpin RNA (shRNA) is a critical concern in RNA interference (RNAi) studies. In the present study, we evaluated biodegradable poly(β-amino ester) (PAE) carrier composed of low molecular weight polyethylenimine and poly(ethylene glycol) for si/shRNA delivery in lung cancer cells. PAE carrier successfully delivered EGFP (enhanced green fluorescence protein) siRNA (siGFP) and silenced EGFP expression. The silencing achieved with PAE carrier was found to be nearly 1.5 times superior and safer than standard PEI25K. Also, our PAE carrier exhibited superior Akt1 shRNA delivery (shAkt) and thereby silenced oncoprotein Akt1 efficiently. PAE–shAkt mediated Akt1 knock-down hindered cancer cell growth in Akt1 specific manner. Superior shAkt delivery and low cytotoxicity of PAE carrier promoted Akt1 knock-down specific apoptosis, while low delivery efficiency and high cytotoxicity of PEI25K carrier mainly exhibited undesirable necrosis. Moreover, basic cancer properties like cell proliferation, malignancy and metastasis were reduced more efficiently using PAE–shAkt system. These findings demonstrated the potential of PAE as an alternative to PEI25K in si/shRNA-based RNAi studies.
Keywords: Non-viral vector; Poly(amino ester); siRNA delivery; Gene silencing; Akt protein; Lung cancer;

Fluorescent magnetic nanohybrids as multimodal imaging agents for human epithelial cancer detection by Jaemoon Yang; Eun-Kyung Lim; Hong Jae Lee; Joseph Park; Sang Cheon Lee; Kwangyeol Lee; Ho-Geun Yoon; Jin-Suck Suh; Yong-Min Huh; Seungjoo Haam (2548-2555).
Cetuximab conjugated fluorescent magnetic nanohybrids (CET-FMNHs) were synthesized for detection of human epithelial cancer via magnetic resonance (MR) and optical imaging. Spherical FMNHs consist of MnFe2O4 magnetic nanocrystals encapsulated in pyrene-labeled PCL-b-PMAA as a surfactant prepared by a nano-emulsion method. FMNHs demonstrated excellent colloidal stability and biocompatibility for biomedical application. Antibody conjugated fluorescent magnetic nanohybrids (CET-FMNHs) served as effective agents for both magnetic resonance (MR) and fluorescence optical imaging of cancer cell lines.
Keywords: Magnetic nanoparticle; Fluorescence; Nanohybrid; Cancer; Cetuximab;

Characterization and optimization of RGD-containing silk blends to support osteoblastic differentiation by Abby W. Morgan; Kristen E. Roskov; Sheng Lin-Gibson; David L. Kaplan; Matthew L. Becker; Carl G. Simon (2556-2563).
The effect of blending two silk proteins, regenerated Bombyx mori fibroin and synthetic spidroin containing RGD, on silk film material structure (β-sheet content) and properties (solubility), as well as on biological response (osteoblast adhesion, proliferation and differentiation) was investigated. Although the elasticity and strength of silks make them attractive candidates for bone, ligament, and cartilage tissue engineering applications, silk proteins generally lack bioactive peptides for enhancing cell functions. Thus, a synthetic spider silk, spidroin, containing two RGD cell adhesive sequences (RGD-spidroin) was engineered. RGD-spidroin was blended with different ratios of fibroin and spun coat into films on glass coverslips. β-Sheet formation, contact angle, surface topography and RGD surface presentation were characterized and correlated with cell behavior. We found that the amount of β-sheet formation was directly related to the RGD-spidroin content of the blends after annealing, with the pure RGD-spidroin demonstrating the highest amount of β-sheet content. The increased β-sheet content improved film stability under culture conditions. A new visualization technique demonstrated that the RGD presentation on the film surface was affected by both the RGD-spidroin content and annealing conditions. It was determined that 10 mass% RGD-spidroin was necessary to improve film stability and to achieve osteoblast attachment and differentiation.
Keywords: Cell adhesion; Cell differentiation; FTIR spectroscopy; Polymer blends; RGD peptide; Silk;