Biomaterials (v.26, #9)

Calendar (I).

Increased osteoblast functions on theta+delta nanofiber alumina by Thomas J. Webster; Elaine L. Hellenmeyer; Rachel L. Price (953-960).
Nanophase materials, or materials with grain sizes less than 100 nm in at least one direction, are promising materials for various implant applications since our tissues are composed of nanometer components (i.e., proteins and/or inorganics). Specifically, bone is comprised of nanostructured hydroxyapatite and collagen fibers which continuously provide an extracellular matrix surface to bone-forming cells (osteoblasts) with a high degree of nanometer roughness. Despite this fact, materials currently utilized for orthopedic implants, whether metallic or ceramic, have constituent grain sizes in the non-biologically inspired micron regime. For this reason, the objective of the present in vitro study was to determine osteoblast functions on one classification of nanomaterials for orthopedic applications: nanofiber alumina. Various crystalline forms of nanofiber alumina were tested in this study. To obtained different crystalline structured nanofiber alumina, boehmite nanofiber alumina was sintered at either 400°C, 600°C, 800°C, 1000°C, or 1200°C for 2 h in air. X-ray diffraction results provided evidence that boehmite nanofiber alumina remained boehmite when sintered at 400°C but changed crystalline phases to gamma, gamma+delta, theta+delta, and alpha when sintered at 600°C, 800°C, 1000°C, and 1200°C, respectively. Moreover, compared to any other alumina formulation tested in this study, osteoblast functions (as measured by alkaline phosphatase activity and calcium deposition) were the greatest on theta+delta crystalline phase nanofiber alumina after 14 days of culture. Boehmite had the next greatest amount of calcium deposition by osteoblasts followed by gamma+delta. Gamma crystalline phase then followed and was greater than alpha crystalline phase nanofiber alumina which promoted osteoblast functions the least of all the compacts with the exception of borosilicate glass (reference substrate). For this reason, this study suggests that theta+delta nanofiber alumina should be further investigated in orthopedic applications.
Keywords: Alumina; Nanophase; Nanofiber; Nanotechnology; Osteoblasts; Orthopedic;

Metalloproteinase and cytokine production by THP-1 macrophages following exposure to chitosan-DNA nanoparticles by Fatiha Chellat; Alexia Grandjean-Laquerriere; Richard Le Naour; Julio Fernandes; L’Hocine Yahia; Moncef Guenounou; Dominique Laurent-Maquin (961-970).
The use of nanoparticles for gene therapy is gaining more and more interest for medical applications. Chitosan is among the candidate polymers that have a potential application as a gene delivery system. Before using chitosan-DNA nanoparticles in vivo, one must study their interaction and cell's behavior. Since macrophages play an important role in inflammatory processes, this study was performed to investigate the effects of chitosan-DNA nanoparticles on human THP-1 cell line. Cytokine (TNF-α, IL-1β, IL-6 and IL-10) and metalloproteinase (MMP-2 and MMP-9) release as well as their inhibitors (TIMP-1 and TIMP-2) were assessed after time course incubation with different amount of nanoparticles. Their secretion was quantified by enzyme-linked immunosorbent assay. Gelatinolytic activity of MMP-2 and MMP-9 was determined by zymography in cell supernatants and lysates.Cytokine secretion was not detected even in the presence of high amount of nanoparticles. On the contrary, the secretion of MMP-9 in cell supernatants increased significantly after 24 and 48 h in comparison with non-treated cells. MMP-2 secretion was augmented only after 48 h for the highest concentrations of nanoparticles (10 and 20 μg/ml DNA content). However, zymography studies showed that the secreted MMPs were in the proactive forms, while the active form of MMP-9, but not MMP-2, was detected in cell lysates when 10 and 20 μg/ml DNA containing nanoparticles were used.In conclusion, exposure of THP-1 macrophages to Ch-DNA nanoparticles did not induce release of proinflammatory cytokines. The presence of active MMP-9 within the macrophages could possibly be related to nanoparticle phagocytosis and degradation rather than to inflammatory reactions.
Keywords: Macrophages; Chitosan-DNA nanoparticles; Metalloproteinases; Cytokines;

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells by Néha Datta; Heidi L. Holtorf; Vassilios I. Sikavitsas; John A. Jansen; Antonios G. Mikos (971-977).
Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze–thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5×105  cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3×106  cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (±13.6) μg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.
Keywords: Bone tissue engineering; Bone; Marrow stromal cells; Extracellular matrix;

Three-dimensional (3D) culture of cancer cell lines has long been advocated as a better model of the malignant phenotype that is most closely related to tumorigenicity in vivo. Moreover, new drug development requires simple in vitro models that resemble the in vivo situation more in order to select active drugs against solid tumours and to decrease the use of experimental animals. A biodegradable, biocompatible and non-toxic polymer chitosan was employed for 3D culture of MCF-7 cell lines. Cells grown on chitosan scaffold produce more lactate from glucose in comparison to that secreted by cells grown on tissue culture plate, thus indicating the suitability of chitosan scaffold as an in vitro model resembling cancer tissue growth in vivo. Cytotoxic effect of tamoxifen at different concentrations was evaluated for MCF-7 breast cancer cell lines grown on tissue culture plate as well as on 3D chitosan scaffold. At a tamoxifen concentration of 10−6  M, 50% reduction in cell growth was observed in tissue culture plate-grown cells where 15% reduction in cell growth was observed when cells were grown in chitosan scaffold. Higher tamoxifen concentrations were required to achieve comparable cytostatic action in 3D culture, supporting the fact that 3D culture is a better model for the cytotoxic evaluation of anticancer drugs in vitro. Carbohydrate metabolism of MCF-7 cells in terms of glucose utilization and lactate production in 3D and monolayer culture were unaffected by tamoxifen treatment. Cathepsin D activity, an autocrine growth factor in breast cancer cells was monitored in all experiments. In 3D culture, addition of tamoxifen promoted cathepsin D secretion but inhibited its uptake by cells. Growth of cells in 3D chitosan scaffold indicated that action of tamoxifen on estrogen positive cancer cells is also mediated through inhibition of cathepsin D uptake from the culture medium.
Keywords: Chitosan scaffold; MCF-7 cell lines; Three dimensional culture; Tamoxifen; Cathepsin D;

The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan by I. Donati; S. Stredanska; G. Silvestrini; A. Vetere; P. Marcon; E. Marsich; P. Mozetic; A. Gamini; S. Paoletti; F. Vittur (987-998).
A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, 1H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5–1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.

Our goal is to fabricate continuous sheets of elastin atop non-biodegradable hydrogels (hylans) containing crosslinked hyaluronan, a glycosaminoglycan. Such elastin–hyaluronan composites may be useful to tissue engineer replacements for the glycosaminoglycan- and elastin-rich layers of the native aortic valve cusp. Neonatal rat aortic smooth muscle cells were cultured atop hylan gels with micro-textured surfaces, and on plastic, and the components of the extracellular matrix (collagen, elastin) were periodically analyzed.The hylan substrates induced the cells to proliferate more rapidly and over longer time periods (∼4 weeks) relative to those cultured on plastic (2–3 weeks). Consequently, at all assay times, the amounts of elastin was derived from the hylan-based cell cultures was 25% or more than that derived from cells cultured on plastic. However, when elastin content was normalized to the cell DNA content, no significant differences were found in the two substrates beyond the first two weeks of culture. Conversely, at culture times greater than 2 weeks, cells cultured atop hylan gels produced amounts of collagen/nanogram of DNA that were ∼56% less than that synthesized by cells cultured on plastic. Cells grown on hylan deposited an unusual matrix layer, rich in elastin, at the hylan-cell interface. This elastin was found to be organized into fenestrated sheets and loose elastin fibers, structures that were also isolated from the elastin matrix of the ventricularis layer of porcine aortic valve cusps. We have thus demonstrated that hylan gels are useful as substrates to induce elastin synthesis in culture to obtain structures that resemble the elastin matrix of the native aortic valve.
Keywords: Hylan; Extracellular matrix; Cell culture; Hydrogel;

Effect of hydration variability on hybrid layer properties of a self-etching versus an acid-etching system by Karen A. Schulze; Sofia A. Oliveira; Robert S. Wilson; Stuart A. Gansky; Grayson W. Marshall; Sally J. Marshall (1011-1018).
The hypothesis tested in this study was that the self-etching system (Clearfil SE Bond, CSE) is less sensitive to surface moisture variability than the system that uses a separate acid-etching step (Single Bond, SB). Eighteen dentin specimens were bonded to composite using CSE or SB. Three different surface moisture conditions per bonding type (overwet, w; dry, d; and visibly moist, n [normal]) were applied prior to bonding dentin to composite. One cross section of each sample was analyzed with lines of nanoindentations crossing perpendicular to the bonding interface. An additional set of bonded samples was fixed and cross sectioned before the hybrid layer thickness was measured in scanning electron microscopy. The nanoindentations revealed significant differences in indentation modulus (E i) and hardness (H) for the hybrid layer comparing SBn, E i=2.7(±1.6); H=0.24(±0.1) GPa with SBd, E i=0.9(±0.7); H=0.07(±0.05) GPa, respectively, while CSE showed no differences among the groups. A significantly greater demineralized zone below the hybrid layer was found for SBd. The hybrid layer was wider for both CSEd and SBd. In conclusion the hypothesis was verified; CSE exhibited no significant changes of hybrid layer properties (E i,H) at different hydration conditions, while SB had significant differences, especially after air-drying.
Keywords: Dentin bonding agent; Composite; Nanoindentation; Adhesion;

Screening for symptomatic metal sensitivity: a prospective study of 92 patients undergoing total knee arthroplasty by Yasuo Niki; Hideo Matsumoto; Toshiro Otani; Taku Yatabe; Makoto Kondo; Fumihiro Yoshimine; Yoshiaki Toyama (1019-1026).
Metal sensitivity (MS) reactions to implant metals represent a rare but well-documented complication following total joint arthroplasty (TJA). Although 20–25% of post-TJA patients develop MS, only a few highly susceptible patients (<1%) exhibit symptoms. Whether surgeons should perform screening for MS is currently a matter of debate. The present study investigated the clinical importance of screening for patients predisposed to symptomatic MS, and the specific metals causing symptomatic MS following total knee arthroplasty (TKA). Between 2000 and 2002, a total of 108 primary TKAs were performed on 92 patients. Preoperatively, all patients underwent modified lymphocyte stimulation test (mLST) to Ni, Co, Cr, and Fe. Of the 92 patients, 24 (26%) displayed positive preoperative responses to at least one metal. Five patients displayed implant metal-related eczema and were all mLST-positive preoperatively, suggesting that screening for symptomatic MS is clinically useful. Two of these underwent revision TKA and thereafter, eczema healed and mLST results changed from positive to negative. All mLST-positive patients were divided into three groups: Group I, patients with eczema; Group II, patients with clear history of MS; and Group III, patients neither eczema nor history of MS. When the type of sensitive metals were compared among the three groups, a significant association between presence of Cr-sensitivity and development of eczema (P<0.05) was identified. No significant association was observed between other metals and development of eczema or history of MS. This indicates that Cr is a potential candidate metal for causing eczema in our TKA series, and Cr-sensitivity may offer a potential predictor for symptomatic MS. The present study indicates that the surgeons should undertake routine preoperative screening for MS, particularly to Cr.
Keywords: Metal sensitivity; Screening; Total knee arthroplasty; Lymphocyte stimulation test;

The interaction between osteoclast-like cells and osteoblasts mediated by nanophase calcium phosphate-hybridized tendons by Hirotaka Mutsuzaki; Masataka Sakane; Atsuo Ito; Hiromi Nakajima; Shinya Hattori; Yutaka Miyanaga; Junzo Tanaka; Naoyuki Ochiai (1027-1034).
We developed a novel technique of hybridizing calcium phosphate (CaP) with bioorganic soft tissue using an alternating soaking process. By this technique, we hybridized CaP with a grafted tendon tissue to bond with a bone tunnel. Tendons were soaked in Ca and NaHPO4 solutions alternately for 10 min. Needle-like CaP crystals 30–50 nm in length including low-crystalline apatite were deposited on and between collagen fibrils from the surface to 200 μm deep in the tendon. In light and transmission electron microscopic images, osteoclast-like cells and osteoblasts appeared on the implanted tendon and osteoid was observed on the tendon surface at 1 week postoperatively. At 2 weeks postoperatively, osteoclast-like cells resolved the tendon by forming Howship's lacuna-like spaces on the surfaces and osteoblasts formed osteoid in these spaces. Direct bonding between the implanted tendon and the newly formed bone was observed. At 3 weeks postoperatively, thick newly formed bone firmly bonded to tendon surface. From these results, we conclude that the tendons prepared by an accelerated CaP hybridization method efficiently enhance osteoclast-like cells and osteoblasts to bond the implanted tendons to newly formed bone.
Keywords: Tendon–bone bonding; Osteoclast; Osteoblast; Nanocrystal; Calcium phosphate hybridization;

A challenge to the conventional wisdom that simultaneous etching and resin infiltration always occurs in self-etch adhesives by Ricardo M. Carvalho; Stefano Chersoni; Roland Frankenberger; David H. Pashley; Carlo Prati; Franklin R. Tay (1035-1042).
This study provided morphological evidence that discrepancies between the depth of demineralisation and the depth of resin infiltration can occur in some mild self-etch adhesives. Sound dentine specimens derived from extracted human third molars were bonded with 5 one-step and 5 two-step self-etch adhesives. One millimeter thick slabs containing the resin–dentine interfaces were immersed in 50 wt% aqueous ammoniacal silver nitrate and processed for TEM examination. A zone of partially etched but uninfiltrated dentine was identified beneath the hybrid layers in the milder versions of both one-step and two-step self-etch adhesives. This zone was characterised by the occurrence of silver deposits along the interfibrillar spaces of mineralised collagen fibrils. The silver infiltrated interfibrillar spaces were clearly identified from the one-step self-etch adhesives Xeno III, iBond, Brush&Bond and the experimental adhesive, and were thinner and only occasionally observed in the two-step self-etch adhesives Clearfil SE Bond and Clearfil Protect Bond. The more aggressive one-step and two-step adhesives that exhibit more abrupt transitions from completely demineralised to mineralised dentin were devoid of these silver-infiltrated interfibrillar spaces beneath the hybrid layers. Incomplete resin infiltration observed in some self-etch adhesives may be caused by the reduced etching potential of the acidic monomers toward the base of hybrid layers, or the presence of acidic but non-polymerisable hydrolytic adhesive components, creating potential sites for the degradation of the bonded created by these self-etch adhesives.
Keywords: Interfibrillar spaces; Self-etch adhesives; Incomplete resin infiltration;

In vivo tissue response to resorbable silica xerogels as controlled-release materials by Shula Radin; Gehan El-Bassyouni; Edward J. Vresilovic; Evert Schepers; Paul Ducheyne (1043-1052).
Biodegradable, controlled-release carrier materials with non-toxic degradation products are valuable for local delivery of biologically active molecules. Previously, it was shown that room-temperature processed silica sol–gels (or xerogels) are porous, resorbable materials that can release molecules of various sizes in a controlled, time dependent manner. Previous in vitro studies also demonstrated benefits of silica xerogels as controlled-release materials for the treatment of bone infections. Herein the tissue and cell response to xerogels is documented using a subacute implantation procedure. The tissue response was correlated to composition, surface properties, resorption rate and incorporation of the antibiotic vancomycin. Ca- and P-free and Ca- and P-containing xerogels, with and without apatite (AP) surface, were used. Xerogels were implanted either as discs in a subcutaneous site, or as granules in the iliac crest of New Zealand white rabbits. The samples with surrounding tissue were retrieved after 2 and 4 weeks of implantation. Silica xerogels implanted either as discs subcutaneously or as granules in the iliac crest showed a favorable tissue response. The granules, either with or without Ca and P content, gradually resorbed over time. The resorption was accompanied by extensive trabecular bone growth and a minimal inflammatory response. Ca- and P-containing granules with an AP-surface layer showed a slower resorption rate and more extensive new bone growth than those without AP layer. Among AP-coated granules, those with incorporated vancomycin showed the most favorable tissue response. The present in vivo data together with prior in vitro data suggest that these xerogels have potential as controlled-release materials for the treatment of bone infections and as carrier materials for a variety of other applications.
Keywords: Controlled-release materials; Silica xerogel; In vivo test; Bioresorption; Biocompatibility;

Biodegradable methoxy poly(ethylene glycol)/poly(ε-caprolactone) (MPEG/PCL) amphiphilic block copolymer nanospheres coupled to folic acid have been designed to target a folate-binding protein that is overexpressed on the surface of many tumoral cells. For this purpose, hydroxy groups terminated on the MPEG/PCL copolymer were converted into primary amino groups, which were used to conjugate with the carboxylic group of folic acid. Nanospheres were prepared by the formation of micelles of the copolymer with or without the anticancer agent paclitaxel. Folate-mediated MPEG/PCL nanospheres were compared with hydroxyl- and amino-terminated nanospheres in terms of their size, surface characteristics, and drug-loading efficiency. Regardless of the type of terminal group, the MPEG/PCL nanospheres showed a narrow size distribution with an average diameter <80 nm without paclitaxel, and an average diameter of 115 nm when loaded with the drug. The results from zeta potential and X-ray photoelectron spectroscopy measurements revealed that the folate molecules were partially exposed, and were expressed on the surface of the nanospheres allowing folate receptor recognition. In in vitro, cytotoxicity tests, the nanospheres loaded with paclitaxel showed a higher cell viability than in cases where paclitaxel was absent. Thus, folate-mediated nanospheres composed of MPEG and PCL are potentially new drug carriers for tumor cell-selective targeting treatments.
Keywords: Folic acid; Nanosphere; Paclitaxel; Poly(ε-caprolactone); Poly(ethylene glycol);

To improve transgene expression of a non-viral gene delivery system, an Epstein–Barr virus (EBV)-based plasmid and cationic emulsion complex was prepared and evaluated. Cationic emulsion was formulated with castor oil, 3-N-(N′,N′-dimethylaminoethane)-carbamoyl cholesterol (DC-Chol) and other co-emulsifiers. An EBV-based plasmid containing the two EBV components, origin of replication (oriP) and EBV nuclear antigen 1 (EBNA-1), was constructed. The physical characteristics of the emulsion and the emulsion/DNA complex were determined. After cells were transfected with cationic emulsion/EBV-based plasmid complex, transfection efficiency and expression pattern were evaluated using green fluorescent protein (GFP) as a reporter. The average particle size and zeta potential of the emulsion itself were 96 nm and +17 mV, respectively. The emulsion showed stable size distribution up to at least one month. With an increase of emulsion to DNA ratio, zeta-potential increased from negative to positive and the particle size decreased to 200–300 nm. The complex was stable against DNase I digestion and showed comparable transfection efficiency with Lipofectin® for several tested cell lines. An enhanced and prolonged gene expression was achieved using EBV-based plasmid and cationic emulsion complex. Combining physically stable emulsion with self-replicating EBV-based plasmid may confer more effective gene expression.
Keywords: Gene expression; Emulsion; Epstein–Barr virus; Episomal plasmid;

Self-organisation of nacre in the shells of Pterioida (Bivalvia: Mollusca) by Antonio G. Checa; Alejandro B. Rodrı́guez-Navarro (1071-1079).
Changes in the morphology and orientation of crystals have been tracked across the nacreous layer in pterioid bivalves, by SEM and X-ray diffraction. Early crystals nucleated on the organic membrane usually covering the outer prismatic layer. They formed polycrystalline aggregates of various shapes, first at prism boundaries and progressively invading the prisms in a centripetal fashion. Their c-axes were perpendicular to the substrate, but the a- and b-axis were variously arranged. As they grew, nacreous crystals became individualized and acquired a common crystallographic orientation also in the ab plane (with the b-axis in the direction of shell growth). Initial lamellae had well delineated growth fronts, which later changed to diffuse. The increasing orientation was explained by a competition model between nacreous plates within the growing lamellae, which was particularly effective in lamellae with delineated fronts. This competition between adjacent crystals could not take place in gastropod nacre because, contrary to the stepped mode of growth of bivalve nacre, vertical stacking predominated. Our model is an alternative to the heteroepitaxial model where the organic matrix supposedly acted as an orienting template, but is nevertheless compatible with the mineral bridge hypothesis that implied epitaxial growth of stacking nacreous tablets.
Keywords: Biomineralisation; Microstructure; Shell; Nacre; Prisms; Bivalves;