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Biomaterials (v.26, #14)

Calendar (pp. i).

Rheometric study of the gelation of chitosan in a hydroalcoholic medium by Alexandra Montembault; Christophe Viton; Alain Domard (pp. 1633-1643).
The formation of chitosan physical hydrogels without any external cross-linking agent was studied. The gelation took place in an acetic acid-water-propanediol solution. The time to reach the gel point was determined by rheometry and gelations from different initial conditions could be compared. The influence of different parameters on gelation such as the polymer concentration, the degree of acetylation (DA) of chitosan and the composition of the initial solvent were investigated. The fractal morphology of the sample was not affected by the composition of the system. The number of junctions per unit volume at the gel point varied only with the initial number of chain entanglements per unit volume. Then, below an initial concentration of 1.5% (w/w), physical chain entanglements were insufficient and more junctions had to be formed to induce gelation. Over this value, only the kinetics allowing to replace entanglements by stable physical junctions played a key parameter. This kinetics was influenced by several parameters such as DA, temperature or the initial proportion water/alcohol. The acetyl groups played an important role in the formation of hydrophobic interactions, mainly responsible for gelation. The study of the influence of the gelation media revealed two critical points at 40% and 70% of water in the initial solvent, probably due to conformational changes and then to different modes of gelation. These physical hydrogels being used for cartilage regeneration, their final rheological properties were studied as a function of their degree of acetylation, the polymer concentration and the solvent composition in the initial solvent. Our results allowed us to define an optimal gelation condition for our application, corresponding to: DA=40%, a proportion water/alcohol of 50/50 and a polymer concentration of 1.5%.

Keywords: Chitin/chitosan; Physical hydrogel; Sol–gel transition; Degree of acetylation; Charge density; Rheology

Laser-induced crystallization of calcium phosphate coatings on polyethylene (PE) by Bastiaan Feddes; Arjen M. Vredenberg; Martin Wehner; Joop C.G. Wolke; John A. Jansen (pp. 1645-1651).
Calcium phosphate (CaP) coatings are used for obtaining a desired biological response. Usually, CaP coatings on metallic substrates are crystallized by annealing at temperatures of at least 400–600°C. For polymeric substrates, this annealing is not possible due to the low melting temperatures. In this work, we present a more suitable method for obtaining crystalline coatings on polymeric substrates, namely laser crystallization. We were successful in obtaining hydroxyapatite coatings on polyethylene. Because of the UV transmission characteristics of the CaP coatings, the use of a low wavelength (157nm) F2 laser was necessary for this. As a result of the laser treatment, the CaP coating broke up into islands. The cracks between the islands became larger and the surface became porous with increasing laser energy. The mechanism behind the formation of this morphology did not become clear. However, the fact that crystalline CaP coatings can be obtained on polymeric substrates in an easy way, possibly allows for the development of new products.

Keywords: Calcium phosphate coating; Crystallinity; Hydroxyapatite coating; Laser; Polyethylene

Highly active, lipase silicone elastomers by Amro M. Ragheb; Michael A. Brook; Michael Hrynyk (pp. 1653-1664).
Lipase Candida rugosa was entrapped in silicone rubber via condensation-cure room temperature vulcanization of silanol-terminated poly(dimethylsiloxane) with tetraethyl orthosilicate as a crosslinker, to give a highly active silicone-enzyme elastomer. The effect on enzyme activity of addition of water and hydrophilic polymeric moieties based on poly(ethylene oxide) 2 was examined, as were crosslinker concentration, enzyme concentration, and elastomer thickness. It was demonstrated that lipase is most active in silicone elastomers and more active in silicone oils than simple hydrocarbons. Crosslink density in these elastomers was not an important factor in the reactivity of the rubber. However, the addition of hydrophilic species prior to elastomer formation decreased the efficiency both of the dispersion of the enzyme and the resulting activity of the elastomer. This effect could be moderated by prior exposure of the lipase to silicone oil. Thus, hydrophobic silicones play a protective/activating role for lipase.

Keywords: Immobilized enzyme; Lipase; Silicone elastomer; Poly(ethylene oxide)

Effect of pH and ionic strength on the reactivity of Bioglass 45S5 by Marta Cerruti; David Greenspan; Kevin Powers (pp. 1665-1674).
Bioglass® 45S5 is a silica-based melt-derived glass, used in medical field as a bone regenerative material because of the deposition of a layer of hydroxy carbonate apatite (HCA) on the surface of the glass when immersed in body fluid.The present paper studies the early steps of reaction of 2-μm sized particles of Bioglass®, in solutions buffered with TRIS at different pH, by means of ICP-ES and FTIR spectroscopy. Only at pH 8 could a total reconstruction of the glass be observed, with the formation of both a silica and a calcium phosphate rich layers. At higher pH, selective dissolution of the glass was hindered by the immediate precipitation of a layer of calcium phosphate, whereas at lower pH a total breakdown of the glass occurred and no calcium phosphate precipitation was noted. The use of the ATR-liquid cell allowed the observation of the reaction in real time, and this showed that the process of silica formation is not separable from cation leaching from the glass, as well as the formation of the calcium phosphate rich layer.

Keywords: Bioactive glass; Micron-size particles; TRIS dissolution; FTIR-ATR; ICP-ES

Photo-polymerized microarchitectural constructs prepared by microstereolithography (μSL) using liquid acrylate-end-capped trimethylene carbonate-based prepolymers by Il Keun Kwon; Takehisa Matsuda (pp. 1675-1684).
Precision microarchitectural constructs made of acrylated trimethylene carbonate (TMC)-based liquid prepolymers were photo-polymerized using a custom-designed microstereolithographic apparatus. In this study, three different photo-polymerizable liquid prepolymers were prepared by the polymerization of TMC with a low molecular weight poly(ethylene glycol) (PEG) (mol. wt. 200 or 1000); designated as PEG200 or PEG1000, respectively or trimethylolpropane (TMP) as an initiator, and subsequently end-capped with an acrylate group. As a result of layer-by-layer photo-irradiation of the prepolymer with a movable ultraviolet light pen driven by computer-aided design, a three-dimensional (3D) micropillar array, a microbank array, a microcone array, and multi-microtunnels formed on a platform plate or a glass plate were precisely fabricated. The PEG-based polymers exhibited a very low cell adhesion potential, whereas the TMP-based hydrophobic polymer exhibited high cell adhesion and proliferation potentials. The microbank array, which consisted of a plate made of the TMP-based polymer and microbanks made of the PEG200-based polymer, caused cell adhesion and proliferation only on the plate. Upon the implantation of microcone arrays under the subcutis of rats, the photo-polymerized construct made of the poorly swellable PEG200-based polymer exhibited only surface erosion and limited drug loading and releasing potentials. On the other hand, the photo-polymerized construct made of the highly swellable PEG1000-based polymer exhibited not only surface erosion but also bulk erosion and high drug loading and releasing potentials. In this paper, we discuss their potential biomedical applications.

Keywords: Microstereolithography (μSL); Acrylated liquid prepolymer; Photo-polymerization; Microarchitectures; Degradation; Drug release

Tissue response to partially in vitro predegraded poly-L-lactide implants by Wim H. De Jong; J. Eelco Bergsma; Joke E. Robinson; Ruud R.M. Bos (pp. 1781-1791).
The in vivo local reaction of as-polymerized poly-L-lactide composed of 96%L-lactide and 4%D-lactide (PLA96) was investigated by histology at 2, 13 and 26 weeks after subcutaneous implantation in rats. In order to simulate possible end stage reactions the PLA96 was also predegraded in vitro until approximately 50% weight loss. The local reaction of predegraded PLA (PLA96168) was compared to the local reaction of polyethylene (PE) and non-predegraded PLA (PLA96).For PE and PLA96 a mild local reaction was observed at all time points consisting of a minimal layer of macrophage like cells with incidentally multinucleated giant cells at the implant interface, surrounded by a mild connective tissue capsule. For PLA96 at weeks 13 and 26 some minimal alterations in terms of degradation and ingrowth of cells was noted. The in vitro incubation (90°C for 168h) of PLA96168 resulted for the thin 0.2mm samples in complete degradation. Predegraded 0.5, 1.0 and 2.0mm PLA96168 samples were implanted and evaluated. The 1.0 and 2.0mm samples could be evaluated for all time points investigated, but some 0.5mm PLA96168 samples were already completely resorbed at week 2 after implantation. In general, responses found for the predegraded PLA96168 at weeks 2, 13 and 26 were similar with a pronounced macrophage infiltrate containing birefringent material, encapsulation of polymer fragments, and the presence of a debris area consisting of polymer and cellular remnants. In lymph nodes foamy macrophages with birefringent material were only observed in lymph nodes draining sites with predegraded PLA96168.Immunohistochemistry was performed for further characterization of the cellular infiltrate. At the implant interface of the non-degrading PE and PLA96, ED1 and OX6 (MHC class II) positive cells were identified. In the capsule macrophage like cells expressed all three macrophage markers ED1, ED2, and ED3. CD4 and CD8 positive cells, indicating T helper and T supressor/cytotoxic cells, respectively, could be observed in low numbers, CD4 more than CD8. Both CD4 and CD8 were occasionally observed within the degrading PLA96168 implant. Polymorphonuclear neutrophilic granulocytes were mainly observed at 2 weeks after implantation.We showed that predegradation could be used as a means to study late tissue reactions to polymers. Complete degradation may be studied with relatively thin implants, but this may lead to rather optimistic interpretation of resorption periods. When materials are intended to be used for screws and/or plates for bone fixation, implants of at least 1.0–2.0mm thickness should be used as these may show a more realistic representation of the resorption characteristics of the material under investigation.

Keywords: PLA implant; Histology; Immunohistochemistry; Predegradation; Biocompatibility

Differential regulation of osteoblasts by substrate microstructural features by O. Zinger; G. Zhao; Z. Schwartz; J. Simpson; M. Wieland; D. Landolt; Barbara Boyan (pp. 1837-1847).
Microtextured titanium implant surfaces enhance bone formation in vivo and osteoblast phenotypic expression in vitro, but the mechanisms are not understood. To determine the roles of specific microarchitectural features in modulating osteoblast behavior, we used Ti surfaces prepared by electrochemical micromachining as substrates for MG63 osteoblast-like cell culture. Cell response was compared to tissue culture plastic, a sand-blasted with large grit and acid-etched surface with defined mixed microtopography (SLA), polished Ti surfaces, and polished surfaces electrochemically machined through a photoresist pattern to produce cavities with 100, 30 and 10μm diameters arranged so that the ratio of the microscopic-scale area of the cavities versus the microscopic-scale area of the flat region between the cavities was equal to 1 or 6. Microstructured disks were acid-etched, producing overall sub-micron-scale roughness (Ra=0.7μm). Cell number, differentiation (alkaline phosphatase; osteocalcin) and local factor levels (TGF-β1; PGE2) varied with microarchitecture. 100μm cavities favored osteoblast attachment and growth, the sub-micron-scale etch enhanced differentiation and TGF-β1 production, whereas PGE2 depended on cavity dimensions but not the sub-micron-scale roughness.

Keywords: Titanium; Microarchitecture; Microstructure; Roughness; Photolithography; Osteoblasts; MG63 cells; PGE; 2; Surface; Electrochemical micromachining

Prevention of the epoxy resin-based root canal sealers-induced cyclooxygenase-2 expression and cytotoxicity of human osteoblastic cells by various antioxidants by Fu-Mei Huang; Yu-Chao Chang (pp. 1849-1855).
Cyclooxygenase-2 (COX-2) is an inducible enzyme believed to be responsible for prostaglandin synthesis at the site of inflammation. Recently, the activation of COX-2 expression may be one of the important pathogenesis of root canal sealers-induced periapical inflammation. However, little is known about whether chemical interaction can modulate the epoxy resin-based root canal sealers-induced cytotoxicity as well as COX-2 expression. The aim of the present study was to investigate the effects of antioxidants catalase, superoxide dismutase (SOD), and N-acetyl-L-cysteine (NAC) on AH26- and Topseal-induced COX-2 mRNA gene and cytotoxicity in human osteoblastic cell line U2OS cells. The results showed that both epoxy resin-based root canal sealers were cytotoxic to U2OS cells in a concentration-dependent manner ( p<0.05). AH26 and Topseal were found to induce COX-2 mRNA gene expression in U2OS cells. The addition of glutathione (GSH) precursor NAC led to decrease the induction of COX-2 mRNA gene expression and cytotoxicity by both AH26 and Topseal ( p<0.05). However, catalase and SOD lacked the ability to prevent AH26-and Topseal-induced cytotoxicity and COX-2 mRNA gene expression (p>0.05). Taken together, the activation of COX-2 mRNA gene expression may be one of the pathogenesis of epoxy resin-based root canal sealers-induced periapical inflammation. In addition, GSH depletion, but not the attack of oxygen free radicals, could be the mechanism for epoxy resin-based root canal sealers-induced cytotoxicity and COX-2 mRNA gene expression. Factors that induce GSH synthesis may appear useful in preventing cell damage mediated by epoxy resin-based root canal sealers.

Keywords: Root canal sealers; Epoxy resin; Cyclooxygenase-2; Glutathione; Antioxidants

The roles of tissue engineering and vascularisation in the development of micro-vascular networks: a review by Ruben Y. Kannan; Henryk J. Salacinski; Kevin Sales; Peter Butler; Alexander M. Seifalian (pp. 1857-1875).
The construction of tissue-engineered devices for medical applications is now possible in vitro using cell culture and bioreactors. Although methods of incorporating them back into the host are available, current constructs depend purely on diffusion which limits their potential. The absence of a vascular network capable of distributing oxygen and other nutrients within the tissue-engineered device is a major limiting factor in creating vascularised artificial tissues. Though bio-hybrid prostheses such as vascular bypass grafts and skin substitutes have already been developed and are being used clinically, the absence of a capillary bed linking the two systems remains the missing link. In this review, the different approaches currently being or that have been applied to vascularise tissues are identified and discussed.

Keywords: Micro-vascular; Tissue engineering; Capillary beds; Tissue vascularisation

The effect of bioactive glass content on synthesis and bioactivity of composite poly (lactic- co-glycolic acid)/bioactive glass substrate for tissue engineering by Jun Yao; Shula Radin; Phoebe S. Leboy; Paul Ducheyne (pp. 1935-1943).
Tissue engineering offers a promising new approach to bone tissue grafting. One material that has received attention in this regard is the polymer poly (lactic- co-glycolic acid) (PLGA). It has the advantage of controllable bioresorption and ease of processing. Another material of interest is bioactive glass (BG), which shows the ability to stimulate osteoblastic differentiation of osteoprogenitor cells. In this study, we reported on the optimal synthesis parameters and the kinetics of formation of calcium phosphate (Ca-P) phase at the surface of PLGA/BG composites. The formation of calcium phosphate layer was confirmed using scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDXA). PLGA-30%BG microspheres based porous scaffolds for bone tissue engineering were examined for their ability to promote osteogenesis of marrow stromal cells (MSC). This porous scaffold supported both MSC proliferation and promoted MSC differentiation into cells expressing the osteoblast phenotype. It therefore demonstrates significant potential as a bone replacement material.

Keywords: PLGA; Bioactive glass; Calcium phosphate; Marrow stromal cells; Tissue engineering

Poly(lactide- co-glycolide) microspheres as a moldable scaffold for cartilage tissue engineering by Nichole R. Mercier; Henry R. Costantino; Mark A. Tracy; Lawrence J. Bonassar (pp. 1945-1952).
This study demonstrates the use of biodegradable poly(lactide- co-glycolide) (PLG) microspheres as a moldable scaffold for cartilage tissue engineering. Chondrocytes were delivered to a cylindrical mold with or without PLG microspheres and cultured in vitro for up to 8 weeks. Cartilagenous tissue formed using chondrocytes and microspheres maintained thickness, shape, and chondrocyte collagen type II phenotype, as indicated by type II collagen staining. The presence of microspheres further enhanced total tissue mass and the amount of glycosaminoglycan that accumulated. Evaluation of microsphere composition demonstrated effects of polymer molecular weight, end group chemistry, and buffer inclusion on tissue-engineered cartilage growth. Higher molecular weight PLG resulted in a larger mass of cartilage-like tissue formed and a higher content of proteoglycans. Cartilage-like tissue formed using microspheres made from low molecular weight and free carboxylic acid end groups did not display increases in tissue mass, yet a modest increased proteoglycan accumulation was detected. Microspheres comprised of PLG with methyl ester end groups yielded a steady increase in tissue mass, with no real increase in matrix accumulation. The microencapsulation of Mg(OH)2 had negative effects on tissue mass and matrix accumulation. The data herein reflect the potential utility of a moldable PLG-chondrocyte system for tissue-engineering applications.

Keywords: Cartilage tissue engineering; Cartilage repair; ECM; Glycosaminoglycan; Microsphere; Scaffold; PLG

Effect of human platelet supernatant on proliferation and matrix synthesis of human articular chondrocytes in monolayer and three-dimensional alginate cultures by C. Gaissmaier; J. Fritz; T. Krackhardt; I. Flesch; W.K. Aicher; N. Ashammakhi (pp. 1953-1960).
Articular cartilage is rich in collagen type II fibres and proteoglycans and is characterized by low cell density. Chondrocytes have specific nutritional requirements and therefore cannot be expanded in vitro without the risk of generating fibroblastoid cells expressing type I collagen. Therefore, various growth conditions were tested for cartilage tissue engineering. Human platelets are a rich source of many growth factors including transforming growth factor- β and platelet-derived growth factor. To investigate the effect of human platelet supernatant (hPS) on chondrocyte proliferation and differentiation, human articular biopsies obtained from three healthy donors. Chondrocytes were isolated and expanded separately in monolayer cultures and seeded in alginate beads in the presence and absence of hPS of 1% or 10% v/v concentration. Transcript levels of genes encoding chondrogenic factors were determined by quantitative reverse transcriptase-polymerase chain reaction. The deposition of types I and II collagen as well as proteoglycan was detected by indirect immunocytochemistry. Addition of hPS activated chondrocyte proliferation in monolayer cultures but induced a dedifferentiation of chondrocytes towards a fibroblast-like phenotype. The expression levels of mRNAs encoding type II collagen, aggrecan and bone morphogenetic protein-2 were reduced in all samples tested. Seeding chondrocytes in alginate beads in the presence of hPS generated a cell population capable of type II collagen expression, even though hPS induced considerable type I collagen expression as well. Differences (1% vs. 10% group, 1% vs. control, 10% vs. control) in the quantitative gene expression of types I and II collagen or of aggrecan were statistically significant (p<0.001). We conclude that addition of hPS may accelerate chondrocyte expansion but can lead to their dedifferentiation.

Keywords: Articular cartilage; Chondrocytes; Platelet supernatant; Tissue engineering

Study of gelatin-containing artificial skin V: fabrication of gelatin scaffolds using a salt-leaching method by Sang Bong Lee; Yong Han Kim; Moo Sang Chong; Seung Hwa Hong; Young Moo Lee (pp. 1961-1968).
Porous gelatin scaffolds were prepared using a salt-leaching method and these were compared to scaffolds fabricated using a freeze-drying method. The salt-leached gelatin scaffolds were easily formed into desired shapes with a uniformly distributed and interconnected pore structure with an average pore size of around 350μm. The mechanical strength and the biodegradation rate of the scaffolds increased with the porosity, and were easily modulated by the addition of salt. After 1 week of in vitro culturing, the fibroblasts in salt-leached scaffolds were mainly attached on the surface of the pores in the scaffold, whereas cells seeded on freeze-dried scaffolds were widely distributed and aggregated on the top and the bottom of the scaffold. After 14d of culturing, the fibroblasts showed a good affinity to, and proliferation on, the gelatin scaffolds without showing any signs of biodegradation. An in vivo study of cultured artificial dermal substitutes showed that an artificial dermis containing the fibroblasts enhanced the re-epithelialization of a full-thickness skin defect when compared to an acellular scaffold after 1 week.

Keywords: Artificial skin; Gelatin; Salt leaching; Scaffold

Peptide-immobilized nanoporous alumina membranes for enhanced osteoblast adhesion by Erin E. Leary Swan; Ketul C. Popat; Tejal A. Desai (pp. 1969-1976).
Bone tissue engineering requires the ability to regulate cell behavior through precise control over substrate topography and surface chemistry. Understanding of the cellular response to micro-environment is essential for biomaterials and tissue engineering research. This research employed alumina with porous features on the nanoscale. These nanoporous alumina surfaces were modified by physically adsorbing vitronectin and covalently immobilizing RGDC peptide to enhance adhesion of osteoblasts, bone-forming cells. X-ray photoelectron spectroscopy (XPS) and scanning electron microscopy (SEM) were used to characterize the modified nanoporous alumina surface. Survey and high-resolution C1s scans suggested the presence of RGDC and vitronectin on the surface and SEM images confirmed the pores were not clogged after modification. Cell adhesion on both unmodified and modified nanoporous alumina was compared using fluorescence microscopy and SEM. RGDC was found to enhance osteoblast adhesion after 1 day of culture and matrix production was visible after 2 days. Cell secreted matrix was absent on unmodified membranes for the same duration. Vitronectin-adsorbed surfaces did not show significant improvement in adhesion over unmodified membranes.

Keywords: Alumina; Bone tissue engineering; Nanotopography; Osteoblast; RGD peptide; XPS

Bone tissue engineering on patterned collagen films: an in vitro study by S. Ber; G. Torun Köse; V. Hasırcı (pp. 1977-1986).
This study aimed at guiding osteoblast cells from rat bone marrow on chemically modified and patterned collagen films to study the influence of patterns on cell guidance. The films were stabilized using different treatment methods including crosslinking with carbodiimide (EDC) and glutaraldehyde, dehydrothermal treatment (DHT), and deposition of calcium phosphate on the collagen membrane.Mesenchymal osteoprogenitor cells were differentiated into osteoblasts and cultured for 7 and 14 days on micropatterned (groove width: 27μm, groove depth: 12μm, ridge width: 2μm) and macropatterned (groove width: 250μm, groove depth: 250μm, ridge width: 100μm) collagen films to study the influence of pattern dimensions on osteoblast alignment and orientation.Fibrinogen was added to the patterned surfaces as a chemical cue to induce osteoblast adhesion. Cell proliferation on collagen films was determined using MTS assay. Deposition of calcium phosphate on the surface of the film increased surface hydrophilicity and roughness and allowed a good cell proliferation. Combined DHT and EDC treatment provided an intermediate wettability, and also promoted cell proliferation. Glutaraldehyde crosslinking was found to lead to the lowest cell proliferation but fibrinogen adsorption on glutaraldehyde treated film surfaces increased the cell proliferation significantly. Macropatterns were first tested for alignment and only microscopy images were enough to see that there is no specific alignment. As a result of this, micropatterned samples with the topography that affect cell alignment and guidance were used. Osteoblast phenotype expression (ALP activity) was observed to be highest in calcium phosphate deposited samples, emphasizing the effect of mineralization on osteoblast differentiation. In general ALP activity per cell was found to decrease from day 7 to day 14 of incubation. SEM and fluorescence microscopy revealed good osteoblast alignment and orientation along the axis of the patterns when micropatterned films were used.This study shows that it is possible to prepare cell carriers suitable for tissue engineering through choice of appropriate surface topography and surface chemistry. Presence of chemical cues and micropatterns on the surface enhance cell orientation and bone formation.

Keywords: Tissue engineering; Micropatterned surfaces; Cell orientation; Osteoblasts; Collagen

De novo engineering of reticular connective tissue in vivo by silk fibroin nonwoven materials by Ilaria Dal Pra; Giuliano Freddi; Jasminka Minic; Anna Chiarini; Ubaldo Armato (pp. 1987-1999).
Biologically tolerated biomaterials are the focus of intense research. In this work, we examined the biocompatibility of three-dimensional (3D) nonwovens of sericin-deprived, Bombyx mori silk fibroin (SF) in β-sheet form implanted into the subcutaneous tissue of C57BL6 mice, using sham-operated mice as controls. Both groups of mice similarly healed with no residual problem. Macroarray analysis showed that an early (day 3) transient expression of macrophage migration inhibitory factor (MIF) mRNA, but not of the mRNAs encoding for 22 additional proinflammatory cytokines, occurred solely at SF-grafted places, where no remarkable infiltration of macrophages or lymphocytes subsequently happened. Even an enduring moderate increase in total cytokeratins without epidermal hyperkeratosis and a transient (days 10–15) upsurge of vimentin occurred exclusively at SF-grafted sites, whose content of collagen type-I, after a delayed (day 15) rise, ultimately fell considerably under that proper of sham-operated places. By day 180, the interstices amid and surfaces of the SF chords, which had not been appreciably biodegraded, were crammed with a newly produced tissue histologically akin to a vascularized reticular connective tissue, while some macrophages but no lymphocytic infiltrates or fibrous capsules occurred in the adjoining tissues. Therefore, SF nonwovens may be excellent candidates for clinical applications since they both enjoy a long-lasting biocompatibility, inducing a quite mild foreign body response, but no fibrosis, and efficiently guide reticular connective tissue engineering.

Keywords: Abbreviations; 3-D; three-dimensional; CT; connective tissue; FBR; foreign body response; GAPDH; glyceraldehyde-3-phosphate dehydrogenase; IC; immunohistochemistry; MIF; macrophage migration inhibitory factor; PGC; polynucleated giant cells; RCT; reticular connective tissue; SF; silk fibroin; vWF; von Willebrand factor; WB; Western immunoblots/-blotting.Silk fibroin; Biocompatibility; Foreign body response; Tissue engineering; Reticular connective tissue; Angiogenesis

Low-density cultures of bovine chondrocytes: effects of scaffold material and culture system by Jerry C. Hu; Kyriacos A. Athanasiou (pp. 2001-2012).
Chondrocytes were seeded on either agarose or polyglycolic acid (PGA) unwoven meshes at 10 million cells/ml of scaffold volume to evaluate the effect that these two biomaterials have on the low-density culture of chondrocytes in a rotating-wall bioreactor. For both static and bioreactor culture, agarose constructs contained more glycosaminoglycan than their PGA counterparts. However, the PGA constructs contained more collagen for both culture conditions when compared to agarose. For the low seeding density of this study, PGA constructs cultured in the bioreactor did not outperform static cultures when comparing collagen content after 8 weeks. The mechanical properties of the PGA constructs also did not improve with culture time. Similar results were observed with the agarose culture, though both static- and bioreactor-culture agarose constructs exhibited increases in aggregate modulus at the end of the culture period. As in PGA culture, chondrocytes cultured in agarose may require a higher density to reap the benefits of the bioreactor environment.

Keywords: Bioreactor; Cartilage tissue engineering; ECM (extracellular matrix); Hydrogel; Mechanical properties; Polyglycolic acid

Effective use of optimized, high-dose (50kGy) gamma irradiation for pathogen inactivation of human bone allografts by Teri A. Grieb; Ren-Yo Forng; Richard E. Stafford; Jack Lin; Jamie Almeida; Simon Bogdansky; Chad Ronholdt; William N. Drohan; Wilson H. Burgess (pp. 2033-2042).
The safety of tissue allografts has come under increased scrutiny due to recent reports of allograft-associated bacterial and viral infections in tissue recipients. We report that 50kGy of gamma irradiation, nearly three times the dose currently used, is an effective pathogen inactivation method when used under optimized conditions that minimize damage to the tissue. Cancellous bone dowels treated with a radioprotectant solution and 50kGy of optimized irradiation had an ultimate compressive strength and modulus of elasticity equal to conventionally irradiated (18kGy) and non-irradiated control bone grafts. We subjected bone dowels treated with this pathogen inactivation method to an in vitro cytotoxicity test using three different mammalian cell lines and concluded that the treated grafts were not cytotoxic. The log reduction of nine pathogens spiked into radioprotectant-treated bone irradiated to 50kGy was also tested. We achieved 4.9logs of inactivation of a model virus for HIV and hepatitis C and 5logs inactivation of a model virus for human parvovirus B-19. Complete inactivation (6.0–9.2logs) of seven clinically relevant microorganisms was demonstrated. The results show that a combination of radioprotectants and optimized, high-dose gamma irradiation is a viable method for producing safer cancellous bone grafts that have the mechanical strength of existing grafts.

Keywords: Bone grafts; Pathogen inactivation; Gamma irradiation; Biomechanical strength; Biocompatibility

Characterisation of resin–dentine interfaces by compressive cyclic loading by Roland Frankenberger; David H. Pashley; Sven M. Reich; Ulrich Lohbauer; Anselm Petschelt; Franklin R. Tay (pp. 2043-2052).
The aims of this in vitro study were to evaluate the ultra-morphological changes in resin–dentine interfaces after different amounts of thermomechanical load (TML), and to determine the corresponding microtensile bond strengths (μTBS). Enamel/dentine discs with a thickness of 2mm were cut from 24 human third molars and bonded with four adhesives involving different adhesion approaches: Syntac (Ivoclar Vivadent; used as multi-step etch-and-rinse adhesive), Clearfil SE Bond (Kuraray; two-step self-etch adhesive), Xeno III (Dentsply DeTrey; mixed all-in-one self-etch primer adhesive system), and iBond (Heraeus Kulzer; non-mixed all-in-one self-etch adhesive). The resin–dentine discs were cut into beams (width 2mm; 2mm dentine, 2mm resin composite) and subsequently subjected to cyclic TML using ascending amounts of mechanical/thermal cycles (20N at 0.5Hz of mechanical load and 5–55°C of thermal cycles: for 0/0, 100/3, 1,000/25, 10,000/250, 100,000/2,500 cycles). Loaded specimens were either cut perpendicularly in order to measure μTBS (n=20; crosshead speed: 1mm/min) or were immersed in an aqueous tracer solution consisting of 50wt% ammoniacal silver nitrate and processed for ultra-morphological nanoleakage examination using transmission electron microscopy (TEM). μTBS were significantly decreased by increasing amounts of TML for all adhesives ( p<0.05). Bond strengths after 0 vs. 100,000 thermomechanical cycles were: Syntac: 41.3/30.1MPa; Clearfil SE Bond 44.8/32.5MPa; Xeno III 27.5/13.7MPa; iBond 27.0/6.2MPa. Relatively early, a certain amount of nanoleakage was observed in all groups by TEM, which was more pronounced for Xeno III and iBond. The incidence of nanoleakage remained stable or was even reduced with increasing load cycles for all adhesives except iBond, where exact failure origins were detected within the adhesive and at the top of the hybrid layer.

Keywords: Dentine; Adhesives; Etch-and-rinse; Self-etch; Thermomechanical loading; Microtensile bond strength; Cyclic loading

Failure analysis of explanted sternal wires by Chun-Ming Shih; Yea-Yang Su; Shing-Jong Lin; C.-C. Chun-Che Shih (pp. 2053-2059).
To classify and understand the mechanisms of surface damages and fracture mechanisms of sternal wires, explanted stainless steel sternal wires were collected from patients with sternal dehiscence following open-heart surgery. Surface alterations and fractured ends of sternal wires were examined and analyzed. Eighty fractured wires extracted from 25 patients from January 1999 to December 2003, with mean implantation interval of 55±149 days (range 5–729 days) after cardiac surgery, were studied by various techniques. The extracted wires were cleaned and the fibrotic tissues were removed. Irregularities and fractured ends were assayed by a scanning electron microscopy. After stereomicroscopy and documentation, the explants were cleaned with 1% sodium hypochlorite to remove the blood and tissues and was followed by cleaned with deionized water and alcohol. The explants were examined by stereomicroscopy, and irregularities on surface and fracture surfaces of sternal wires were assayed by scanning electron microscopy, energy dispersive X-ray analysis (EDAX) and X-ray mapping. The explants with surrounding fibrotic tissue were stained and examined with stereomicroscopy and transmission electronic microscopy. Corrosion pits were found on the surface of explanted sternal wires. EDAX and X-ray mapping examinations revealed diminution of nickel concentration in the severely corroded pits on sternal wires. A feature of transgranular cracking was observed for stress corrosion cracking and striation character for typical corrosion fatigue was also identified. TEM examination of tissue showed the metallic particles in phagolysosomes of macrophages inside the surrounding sternal tissue. The synergic effect of hostile environment and the stress could be the precursors of failures for sternal wires.

Keywords: Corrosion fatigue; Stainless steel sternal wires; Sternal dehiscence

Epithelial internalization of superparamagnetic nanoparticles and response to external magnetic field by Kenneth Dormer; Charles Seeney; Kevin Lewelling; Guoda Lian; Donald Gibson; Matthew Johnson (pp. 2061-2072).
Superparamagnetic magnetite nanoparticles (MNP) coated with silica were synthesized and chronically implanted into the middle ear epithelial tissues of a guinea pig model (n=16) for the generation of force by an external magnetic field. In vivo limitations of biocompatibility include particle morphology, size distribution, composition and mode of internalization. Synthesis of MNP was performed using a modified precipitation technique and they were characterized by transmission electron microscopy, X-ray diffractometry and energy dispersive spectroscopy, which verified size distribution, composition and silica encapsulation. The mechanism for internalizing 162.3nm diameter MNP was likely endocytosis, enhanced by magnetically force. Using sterile technique, middle ear epithelia of tympanic membrane or ossicles was exposed and a suspension of particles with fluoroscein isothiocyanate (FITC) label applied to the surface. A rare earth, NdFeBo magnet (0.35T) placed under the animal, was used to pull the MNP into the tissue. After 8 days, following euthanasia, tissues were harvested and confocal scanning laser interferometry was used to verify intracellular MNP. Displacements of the osscicular chain in response to an external sinusoidal electromagnetic field were also measured using laser Doppler interferometry. We showed for the first time a physiologically relevant, biomechanical function, produced by MNP responding to a magnetic field.

Keywords: Nanoparticles; Middle ear; Biomechanics; Hearing; Endocytosis

Self-gelling hydrogels based on oppositely charged dextran microspheres by Sophie R. Van Tomme; Mies J. van Steenbergen; Stefaan C. De Smedt; Cornelus F. van Nostrum; Wim E. Hennink (pp. 2129-2135).
This paper presents a novel self-gelling hydrogel potentially suitable for controlled drug delivery and tissue engineering. The macroscopic gels are obtained by mixing dispersions of oppositely charged crosslinked dextran microspheres. These microspheres in turn were prepared by crosslinking of dextran derivatized with hydroxyethyl methacrylate emulsified in an aqueous poly(ethylene glycol) solution. Negatively or positively charged microspheres were obtained by addition of methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA) to the polymerization mixture. Rheological analysis showed that instantaneous gelation occurred when equal volumes of oppositely charged microspheres, dispersed in buffer solutions of pH 7, were mixed. The shear modulus of the networks could be tailored from 30 to 6500Pa by varying the water content of the system. Moreover, controlled strain and creep experiments showed that the formed networks were mainly elastic. Importantly for application of these systems, e.g. as controlled matrix of pharmaceutically active proteins, it was demonstrated that the hydrogel system has a reversible yield point, meaning that above a certain applied stress, the system starts to flow, whereas when the stress is removed, gel formation occurred. Further it was shown that the network structure could be broken by either a low pH or a high ionic strength of the medium. This demonstrates that the networks, formed at pH 7 and at low ionic strength, are held together by ionic interactions between the oppositely charged dextran microspheres. This system holds promise as injectable gels that are suitable for drug delivery and tissue engineering applications.

Keywords: Injectable hydrogels; Dextran microspheres; Ionic interactions; Viscoelasticity; Drug delivery; Tissue engineering

Size and temperature effects on poly(lactic- co-glycolic acid) degradation and microreservoir device performance by A.C.R. Amy C. Richards Grayson; Michael J. Cima; Robert Langer (pp. 2137-2145).
The component materials of controlled-release drug delivery systems are often selected based on their degradation rates. The release time of a drug from a system will strongly depend on the degradation rates of the component polymers. We have observed that some poly(lactic- co-glycolic acid) polymers (PLGA) exhibit degradation rates that depend on the size of the polymer object and the temperature of the surrounding environment. In vitro degradation studies of four different PLGA polymers showed that 150μm thick membranes degraded more rapidly than 50μm thick membranes, as characterized by gel permeation chromatography and mass loss measurements. Faster degradation was observed at 37°C than 25°C, and when the saline media was not refreshed. A biodegradable polymeric microreservoir device that we have developed relies on the degradation of polymeric membranes to deliver pulses of molecules from reservoirs on the device. Earlier molecular release was seen from devices having thicker PLGA membranes. Comparison of an in vitro release study from these devices with the degradation study suggests that reservoir membranes rupture and drug release occurs when a membrane threshold molecular weight of 5000–15000 is reached.

Keywords: Controlled drug release; Degradation; Hydrolysis; Poly(lactic acid); Poly(glycolic acid)

The use of chitosan as a condensing agent to enhance emulsion-mediated gene transfer by Mi-Kyung Lee; Soo-Kyung Chun; Woo-Jeong Choi; Jin-Ki Kim; Sung-Hee Choi; Adele Kim; Kwunchit Oungbho; Jeong-Sook Park; Woong Shick Ahn; Chong-Kook Kim (pp. 2147-2156).
Previously we have formulated a new cationic emulsion, composed of 3 β [ N-( N′, N′-dimethylaminoethane) carbamoyl] cholesterol and dioleoylphosphatidyl ethanolamine, castor oil and Tween 80, and it efficiently delivered plasmid DNA into various cancer cells with low toxicity. Chitosan is a natural cationic polysaccharide and is able to form polyelectrolyte complexes with DNA, in which the DNA is condensed and protected against nuclease degradation. Based on these facts, chitosan was used as a condensing agent to enhance the transfection efficiency of cationic emulsion-mediated gene delivery vehicle. The particle size, zeta potential and transmission electron micrographs of DNA/emulsion complexes were observed before and after condensation by chitosan. In vitro transfection efficiency of naked or precondensed DNA/emulsion (pcDNA/E) complexes was investigated in human hepatoma cells (HepG2) using flow cytometer, confocal microscope and western blot. In addition, in vivo gene transfer was also evaluated as GFP mRNA expression by reverse transcriptase-polymerase chain reaction. The size of transfection complexes was reduced after the condensation of DNA by chitosan. Moreover, when the pcDNA/E complexes were administered into the mice, the GFP mRNA expression was prolonged in liver and lung until day 6. These results suggest that the use of chitosan enhance the in vitro transfection efficiency and extend in vivo gene transfer.

Keywords: Gene therapy; Non-viral vectors; Chitosan; Emulsion; Precondensed DNA

DNA encapsulated magnesium and manganous phosphate nanoparticles: potential non-viral vectors for gene delivery by Gajadhar Bhakta; Susmita Mitra; Amarnath Maitra (pp. 2157-2163).
Nanoparticles of Mg and Mn (II) phosphates encapsulating pDNA were prepared. The sizes of these DNA loaded particles in aqueous dispersion were about 100–130nm diameter, and they aggregated with the progression of time. Although magnesium phosphate nanoparticles were crystalline, the manganous phosphate nanoparticles were found to be amorphous in nature. Nanoparticle dissolution and pDNA release were studied using atomic absorption spectroscopy and gel electrophoresis experiments. These inorganic phosphate nanoparticles dissolved in mild acidic pH (∼5) releasing pDNA indicating that DNA release in the endosomal compartment is possible. In vitro transfection in HeLa cells demonstrated that while magnesium phosphate nanoparticles showed 100% efficiency, manganous phosphate nanoparticles exhibited about 85% transfection efficiency compared to that of ‘polyfect’, as control.

Keywords: Magnesium phosphate; Manganous phosphate; Nanoparticles; Non-viral vectors; Gene delivery; Transfection efficiency

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