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Biomaterials (v.28, #22)

Editorial board (pp. ifc).

The effect of electrochemically simulated titanium cathodic corrosion products on ROS production and metabolic activity of osteoblasts and monocytes/macrophages by Marie Kalbacova; Sophie Roessler; Ute Hempel; Roman Tsaryk; Kirsten Peters; Dieter Scharnweber; James C. Kirkpatrick; Peter Dieter (pp. 3263-3272).
Nowadays aseptic loosening is the most common cause of orthopaedic implant failure. Some of its reasons have already been described up to now; however, others remain still hypothetical. Besides the inflammatory response to wear particles originating at different sources, the role of reactive oxygen species as products of cellular reactions and/or as a result of the process of corrosion of an implant leading to implant failure has recently been discussed too. In the present study, we used a galvanostatic polarization to simulate the cathodic partial reaction of the corrosion process at a titanium alloy surface. With respect to cells occurring at the interface of a metal implant, the behaviour of osteoblasts and monocytes/macrophages was investigated. It has been found that cathodic polarization of Ti6Al4V induces an increase in the level of intracellular reactive oxygen species as well as suppressing the metabolic activity of cells in a dose-dependent manner. This is in agreement with the results obtained with cells after external addition of hydrogen peroxide as another kind of oxidative stress. In both approaches, monocytes/macrophages show a higher tolerance to oxidative stress than osteoblasts. It could be concluded that the electrochemical setup developed induced intracellular changes occurring during oxidative stress and it could be used for future detailed analysis of the consequences of corrosion processes for cellular reactions.

Keywords: Corrosion; Electrochemistry; Oxidative stress; Titanium alloy; Osteoblast; Monocyte

Effects of surface wettability and contact time on protein adhesion to biomaterial surfaces by Li-Chong Xu; C.A. Christopher A. Siedlecki (pp. 3273-3283).
Atomic force microscopy (AFM) was used to directly measure the adhesion forces between three test proteins and low density polyethylene (LDPE) surfaces treated by glow discharge plasma to yield various levels of water wettability. The adhesion of proteins to the LDPE substrates showed a step dependence on the wettability of surfaces as measured by the water contact angle ( θ). For LDPE surfaces with θ>∼60–65°, stronger adhesion forces were observed for bovine serum albumin, fibrinogen and human FXII than for the surfaces with θ<60°. Smaller adhesion forces were observed for FXII than for the other two proteins on all surfaces although trends were identical. Increasing the contact time from 0 to 50s for each protein–surface combination increased the adhesion force regardless of surface wettability. Time varying adhesion data was fit to an exponential model and free energies of protein unfolding were calculated. This data, viewed in light of previously published studies, suggests a 2-step model of protein denaturation, an early stage on the order of seconds to minutes where the outer surface of the protein interacts with the substrate and a second stage involving movement of hydrophobic amino acids from the protein core to the protein/surface interface. Impact statement: The work described in this manuscript shows a stark transition between protein adherent and protein non-adherent materials in the range of water contact angles 60–65°, consistent with known changes in protein adsorption and activity. Time-dependent changes in adhesion force were used to calculate unfolding energies relating to protein–surface interactions. This analysis provides justification for a 2-step model of protein denaturation on surfaces.

Keywords: AFM; Protein; Adhesion; Wettability

Biodegradation of unsaturated poly(ester-amide)s and their hydrogels by Kai Guo; C.-C. Chih-Chang Chu (pp. 3284-3294).
The biodegradability of both unsaturated (UPEA) and saturated (SPEA) poly(ester-amide)s and a series of hydrogels (UPEA-G) fabricated from UPEA and poly(ethylene glycol) diacrylate (PEG-DA) was examined as a function of PEA chemical structures in both phosphate buffered saline (PBS) and α-chymotrypsin solutions. Based on the weight loss data, α-chymotrypsin had a much more profound effect on the hydrolyses of UPEA, SPEA polymers (up to 32% weight loss on day 1 for FPBe) and UPEA-G hydrogels (up to 32% weight loss on day 31 for FPBe-G28) than a PBS buffer (less than 10% for polymers and 16% for hydrogels). The changes in elastic moduli and the interior morphology of the hydrogels in both PBS buffer and α-chymotrypsin solutions were also monitored for 2 months, and the hydrogels’ crosslinking density ( ne) and molecular weight between crosslinks ( Mc) before and after biodegradation were then examined as a function of biodegradation time, enzyme concentration, and different chemical structure of precursors. The differences in biodegradation rates among PEA polymer and UPEA-G hydrogels are ascribed to differences in hydrophilicity and saturated or unsaturated structure of the polymers and hydrogel precursors. Our results showed that, by changing the concentration of α-chymotrypsin, the type of UPEA precursors and their feed ratio, the UPEA-G hydrogels could have controllable biodegradability, which is quite desirable for a wide range of biomedical and pharmaceutical applications.

Keywords: Poly(ester-amide)s; Biodegradable polymers; Hydrogels; Biodegradability; Enzymes

The safety and efficacy of an injectable bone substitute in dental sockets demonstrated in a human clinical trial by Pierre Weiss; Pierre Layrolle; L.P. Léon Philippe Clergeau; B. Bénédicte Enckel; Paul Pilet; Yves Amouriq; Guy Daculsi; Bernard Giumelli (pp. 3295-3305).
This study is the first report of a clinical evaluation of an injectable bone substitute (IBS). This IBS was prepared by suspending biphasic calcium phosphate (BCP) particles with diameters ranging between 80 and 200μm in a water-soluble cellulose polymer carrier phase. It was used for filling bone defects after tooth extractions in 11 patients. The first objective of the study was to investigate the safety of the filler material. The second objective was to investigate the efficacy of the material for filling human tooth sockets and preventing alveolar bone loss. Radiographic density measurements of the surgical sites gradually increased to those of the surrounding host bone. Three years after surgery, small biopsies of the implanted areas were harvested and analyzed by using micro-computed tomography, non-decalcified histology and histomorphometry. The BCP granules appeared in direct contact with mineralized bone tissue, thereby supporting bone growth. A gradual substitution of the filler by bone tissue was observed thus preserving the height of the alveolar bone crest.

Keywords: Calcium phosphate; Biomaterial; Injectable bone substitute; Human trial; Bone healing

Effects of calcium ion incorporation on bone healing of Ti6Al4V alloy implants in rabbit tibiae by J.-W. Jin-Woo Park; K.-B. Kwang-Bum Park; J.-Y. Jo-Young Suh (pp. 3306-3313).
The biocompatibility of calcium ion (Ca)-incorporated Ti6Al4V alloy implants, produced by hydrothermal treatment using a Ca-containing solution, was investigated. The surface characteristics were evaluated by scanning electron microscopy, thin-film X-ray diffractometry, Auger electron spectroscopy, and stylus profilometry. The viability of MC3T3-E1 cells on Ca-incorporated machined Ti6Al4V surfaces with different oxide thicknesses was compared with that on untreated machined Ti6Al4V surfaces with MTT assay. The osteoconductivity of the Ca-incorporated Ti6Al4V implants was evaluated by removal torque testing and histomorphometric analysis after 6 weeks of implantation in rabbit tibiae. Our results show that hydrothermal treatment with a Ca-containing solution produced a crystalline CaTiO3 layer on Ti6Al4V surfaces, and calcium ions were gradually incorporated throughout the oxide layer. After immersion in Hank's balanced salt solution, a considerable apatite deposition was observed on all surfaces of the Ca-incorporated samples. Significant increases in cell viability ( p<0.001), removal torque forces, and bone-to-implant contact values ( p<0.05) were observed for Ca-incorporated Ti6Al4V implants compared with those for untreated Ti6Al4V implants.

Keywords: Titanium alloy; Calcium; Biocompatibility; Osseointegration; Histomorphometry; Cell viability

The performance of a bone-derived scaffold material in the repair of critical bone defects in a rhesus monkey model by Huiqi Xie; Fuchun Yang; Li Deng; Jingcong Luo; Tingwu Qin; Xiuqun Li; G.-Q. Guang-Qian Zhou; Zhiming Yang (pp. 3314-3324).
The efficacy and safety of a material derived from human bones in the repair of critical segmental bone defects are evaluated in a rhesus monkey model. Frozen human bones were chemically and physically processed into a partially demineralized and deproteinized material in blocks. The complete tissue-engineered (TE) bone was constructed of the material preseeded with allogeneic bone marrow mesenchymal stem cells (MSCs). The material alone and the TE bone were, respectively, implanted to bridge 2.5cm-long critical defects in right and left radii of 15 monkeys. At weeks 1, 2, 3, 6 and 12 post -implantation, the grafts were collected from three animals and assessed for the local expression of osteogenic markers, histological and roentgenographic features, and immune reactions. It was shown that defects were well repaired with both treatments whereas the bone defects in 2 additional untreated animals remained the same size after 12 weeks. In radii implanted with the TE bones, the repair processes were approximately 3 weeks faster and new bones were formed in a multipoint way. There was neither observable toxic effect nor overt immune rejection in any animals. Taken together, these observations suggest that the TE bone blocks composited of the allogeneic or xenogeneic bone-derived scaffold and allogeneic MSCs may provide an ideal method for repairing large segmental bone defects.

Keywords: Bone-derived material; Tissue-engineered bone; Bone marrow mesenchymal stem cells; Rhesus monkey; Critical bone defects

Controlling cell adhesion to surfaces via associating bioactive triblock proteins by Stephen E. Fischer; Xingyu Liu; H.-Q. Hai-Quan Mao; James L. Harden (pp. 3325-3337).
A surface functionalization strategy that produces substrates with well-controlled ligand density is critical to investigating the role of cell–substrate interactions in regulating cell adhesion, viability, migration, proliferation and differentiation. Towards this end, we have designed and synthesized a triblock protein, CRC, comprising a polyelectrolyte domain flanked by two amphiphilic leucine zipper domains. The amphiphilic end domains of CRC adsorb onto surfaces and preferentially associate into trimeric aggregates, forming a hydrogel coating layer. Under serum-free conditions, the CRC coating was found to render both 2D substrates and 3D scaffolds non-adhesive to cells. A RGDS sequence was inserted in the middle domain of CRC (generating the protein CRC-RGDS) and found to introduce cell-binding activity. Incorporation of the RGDS sequence did not significantly impact the surface activity of CRC, allowing us to titrate the RGDS surface density simply by adjusting the relative ratios of the two proteins. Ligand density dependent cell–substrate interactions were demonstrated in human foreskin fibroblasts, human umbilical vein endothelial cells, and rat neural stem cells. The versatility to functionalize a range of different substrate surfaces, combined with the ease of controlling surface ligand density, makes these triblock proteins an attractive tool for developing cell-specific surface coatings with tailored biofunctional attributes.

Keywords: Surface functionalization; Cell adhesion; Self-assembly; Protein hydrogels; Cell–materials interactions

Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering by Huanan Wang; Yubao Li; Yi Zuo; Jihua Li; Sansi Ma; Lin Cheng (pp. 3338-3348).
In this study, we prepared nano-hydroxyapatite/polyamide (n-HA/PA) composite scaffolds utilizing thermally induced phase inversion processing technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Mesenchymal stem cells (MSCs) derived from bone marrow of neonatal rabbits were cultured, expanded and seeded on n-HA/PA scaffolds. The MSC/scaffold constructs were cultured for up to 7 days and the adhesion, proliferation and differentiation of MSCs into osteoblastic phenotype were determined using MTT assay, alkaline phosphatase (ALP) activity and collagen type I (COL I) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negative effects on the MSCs in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds and MSC/scaffold constructs were implanted in rabbit mandibles and studied histologically and microradiographically. The results show that n-HA/PA composite scaffolds exhibit good biocompatibility and extensive osteoconductivity with host bone. Moreover, the introduction of MSCs to the scaffolds dramatically enhanced the efficiency of new bone formation, especially at the initial stage after implantation. In long term (more than 12 weeks implantation), however, the pure scaffolds show as good biocompatibility and osteogenesis as the hybrid ones. All these results indicate that the scaffolds fulfill the basic requirements of bone tissue engineering scaffold, and have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery.

Keywords: Bone tissue engineering; Scaffolds; Nano-hydroxyapatite; Polyamide; Mesenchymal stem cells; Osteogenesis

Glycoconjugated peptide dendrimers-based nanoparticulate system for the delivery of chloroquine phosphate by Payal Agrawal; Umesh Gupta; N.K. Jain (pp. 3349-3359).
Dendrimers consisting of different molecules of metabolic pathways such as amino acids can greatly reduce the toxicity associated with amine-terminated dendrimers e.g. polyamidoamine (PAMAM) and polypropylene imine (PPI) dendrimers. In the present study, poly-l-lysine dendrimers having polyethyleneglycol (PEG-1000) as core, were synthesized upto fourth generation. Dendrimers were synthesized by alternating protection and deprotection steps ofl-lysine by di-BOC (di-tertiary butyl pyrocarbonate) till the formation of 4.0G peptide dendrimer took place.d-galactose was selected as model sugar for peripheral conjugation (coating) of these peptide dendrimer. The complete formation of uncoated and galactose-coated poly-l-lysine dendrimers was characterized by transmission electron microscopy (TEM), IR, NMR and MALDI TOF mass spectroscopic studies. Chloroquine phosphate (CP)-loaded uncoated and coated dendrimers were evaluated for in vitro drug release rate, hemolytic toxicity and stability studies. Ex vivo cellular uptake studies of uncoated and coated drug dendrimer formulations in macrophages revealed almost 5 times reduced phagocytosis due to galactose coating ( p<0.0001). In vitro–in vivo release behavior indicated possibilities of galactose-coated drug dendrimers formulation in controlled drug delivery of CP. Galactose coated formulations drastically reduced hemolytic toxicity compared to uncoated poly-l-lysine formulation as well as plain drug. Hematological data suggests that galactose-coated formulations are less immunogenic compared to uncoated formulations. Finally, it can be concluded that galactose-coated polylysine dendrimers can be utilized for controlled delivery of CP more safely compared to its uncoated formulation both in vitro and in vivo.

Keywords: Chloroquine phosphate; Controlled delivery; Peptide dendrimer; Glycoconjugation

Water-soluble chitosan-based antisense oligodeoxynucleotide of interleukin-5 for treatment of allergic rhinitis by S.T. Sung Tae Kim; C.-K. Chong-Kook Kim (pp. 3360-3368).
Interleukin (IL)-5 produced by allergen specific T cells is a major cytokine in the allergic inflammation such as allergic rhinitis (AR). To inhibit the production of IL-5, water-soluble chitosan (WSC)-based IL-5 antisense oligodeoxynucleotide (AS-ODN) complex was generated. WSC, a biocompatible cationic polymer, was used as a non-viral vector for the improvement of stability and transfection efficiency. After condensation IL-5 AS-ODN with WSC, the size, morphology and zeta potential analysis of IL-5 AS-ODN/WSC complexes were performed. The protective effect of complex was also observed against the enzymatic degradation. In vitro transfection efficiency into H1299 epithelial cells was investigated by flow cytometer and inhibition effect of IL-5 levels was also evaluated in D10.G4.1 cells. In the murine model with AR, the IL-5 and IgE levels closely related to the allergic inflammation were significantly reduced after the intranasal administration of IL-5 AS-ODN/WSC complexes. Based on these results, the condensation with WSC improved the physicochemical stability and transfection efficiency of IL-5 AS-ODN/WSC complex. Our results suggest that AS therapy using IL-5 AS-ODN/WSC complex can be an effective strategy in regulating IL-5 and may be applied to the treatment of allergic disorder related to IL-5.

Keywords: Interleukin-5; Water-soluble chitosan; Antisense oligodeoxynucleotide; Allergic rhinitis

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