Biomaterials (v.26, #11)

Calendar (I).

Hydroxyapatite (HA) plays an important role in orthopedics and dentistry due to its excellent bioactivity. However, the thermal decomposition and the poor corrosion resistance in an acid environment have restricted the applications of HA. In this study, several fluorine-substituted hydroxyapatite (FHA) ceramics with the general chemical formula Ca10(PO4)6(OH)2−2x F2x , where x=0.0,0.2,0.4,0.6,0.8,1.0, were prepared. Thermogravimetric analysis in the temperature range from 25°C to 1400°C showed that the FHA ceramics with x>0.4 had remarkably improved thermal stability as compared to pure HA. X-ray diffraction of the FHA ceramics sintered at 1300°C for 1 h further confirmed the thermal stability. Dilatometer analysis showed that the fluorine addition substantially increased the onset sintering temperature of the FHA ceramics. Density measurements showed that the fluorine addition into the HA matrices slightly retarded the densification of the FHA ceramics. Corrosion testing on the polished surfaces of the FHA ceramics using a 2.5 wt% citric acid solution indicated that the FHA ceramics with x⩾0.4 had substantially improved corrosion resistance.
Keywords: Hydroxyapatite; Fluoroapatite; Stability; Decomposition; Sintering;

Variable cytocompatibility of six cell lines with photoinitiators used for polymerizing hydrogels and cell encapsulation by Christopher G. Williams; Athar N. Malik; Tae Kyun Kim; Paul N. Manson; Jennifer H. Elisseeff (1211-1218).
The development of biocompatible photopolymerizing polymers for biomedical and tissue engineering applications has the potential to reduce the invasiveness and cost of biomaterial implants designed to repair or augment tissues. However, more information is needed about the cellular toxicity of the compounds and initiators used in these systems. The current study evaluates the cellular toxicity of three ultraviolet sensitive photoinitiators on six different cell populations that are used for engineering numerous tissues. The photoinitiator 2-hydroxy-1-[4-(hydroxyethoxy)phenyl]-2-methyl-1-propanone (Irgacure 2959) caused minimal toxicity (cell death) over a broad range of mammalian cell types and species. It was also demonstrated that different cell types have variable responses to identical concentrations of the same photoinitiator. While inherent differences in the cell lines may contribute to the variable cytotoxicity, a correlation between cellular proliferation rate (population doubling time) and increased cytotoxicity of the photoinitiator was observed. Cell lines that divided more quickly were more sensitive to photoinitiator-induced cell death. In summary, the photoinitiator Irgacure 2959 is well tolerated by many cell types over a range of mammalian species. Cell photoencapsulation strategies may be optimized to improve cell survival by manipulating proliferation rate.
Keywords: Photopolymerization; Cytotoxicity; Hydrogel; Cell encapsulation; Cell proliferation; Free radical;

Surface properties and cell response of low metal ion release Ti-6Al-7Nb alloy after multi-step chemical and thermal treatments by Silvia Spriano; Michela Bosetti; Marco Bronzoni; Enrica Vernè; Giovanni Maina; Valerio Bergo; Mario Cannas (1219-1229).
Ti-6Al-7Nb samples treated by innovative multi-step chemical and thermal processes were characterized in order to evaluate their surface properties and cell interaction. The main object was to asses if the treatments were effective in order to obtain a surface presenting at the same time bone-like apatite induction ability, low metal ion release, good cell response and high protein binding. The morphology, crystallographic structure, porosity and wettability of the treated materials were investigated, as well as their interaction with simulated body fluid during soaking for different times. Cytotoxicity, protein adsorption tests and in vitro fibroblast and osteoblast-like cell cultures were also performed.
Keywords: Titanium alloys; Surface modification; Osteointegration; Ion release; Protein adsorption; Cell culture;

Coil dimensions of the mussel adhesive protein Mefp-1 by Sander Haemers; Mieke C. van der Leeden; Gert Frens (1231-1236).
To obtain a better understanding of factors controlling cross-linking rates of Mussel adhesive proteins, we study the conformation of the Mussel Adhesive Protein Mefp-1. The dimensions of Mefp-1 in solution are determined by dynamic light scattering. Under physiological conditions, the hydrodynamic radius R H of Mefp-1 is found to be 10.5±1.1 nm. Measured Mefp-1 dimensions are compared with theoretical dimensions of Mefp-1 in random coil conformations. We have strong indications that Mefp-1, under dilute and physiological conditions, has a self-avoiding random walk conformation with helix-like deca-peptide segments. With a number of segments of approximately 90, the segment length is found to be 2.7 nm.
Keywords: Mussel adhesive protein; Mefp-1; Conformation; Cross-linking; Dynamic light scattering;

Tannic acid treatment enhances biostability and reduces calcification of glutaraldehyde fixed aortic wall by Jason C. Isenburg; Dan T. Simionescu; Naren R. Vyavahare (1237-1245).
Progressive degeneration and calcification of glutaraldehyde (Glut) fixed tissues used in cardiovascular surgery restrict their long-term clinical performance. This limited biological stability may be attributable to the inability of Glut to adequately protect certain tissue components such as elastin from enzymatic attack. The aim of our studies was to develop novel tissue-processing techniques targeted specifically at elastin stabilization by using tannic acid (TA), a plant polyphenol capable of protecting elastin from digestion by specific enzymes. In present studies we demonstrated that Glut does not adequately protect porcine aorta from elastase-mediated degradation in vitro. The addition of TA to the Glut fixation process increased the stability of Glut-fixed aorta to elastase digestion by 15-fold and also decreased calcification in the rat subdermal model by 66%. TA was found to be chemically compatible with Glut fixation and did not hinder collagen crosslinking as shown by minor changes in thermal denaturation temperatures, resistance to collagenase and mechanical properties. In vitro and in vivo studies also revealed that TA binding to aortic wall was stable over an extended period of time. TA-mediated elastin stabilization in Glut-fixed cardiovascular implants may significantly extend the clinical durability of these tissue replacements.
Keywords: Elastin; Degradation; Stabilization; Fixation; Tannins; Calcification;

Encapsulation of chondrocytes in injectable alkali-treated collagen gels prepared using poly(ethylene glycol)-based 4-armed star polymer by Tetsushi Taguchi; Liming Xu; Hisatoshi Kobayashi; Akiyoshi Taniguchi; Kazunori Kataoka; Junzo Tanaka (1247-1252).
An in situ gel system was developed to encapsulate chondrocytes under physiological conditions using an alkali-treated collagen (AlCol) and pentaerythritol poly(ethylene glycol) ether tetrasuccinimidyl glutarate (4S-PEG) as a crosslinker. AlCol gels were obtained at crosslinker concentrations from 0.1 to 3.0 mm. Chondrocytes were encapsulated and dispersed homogeneously in AlCol gels. Results of MTT staining showed that cells survived after encapsulation in AlCol gels. Biochemical analysis demonstrated that DNA content in AlCol gels was constant after 3 weeks. Glycosaminoglycan content and mRNA expression of type II collagen and aggrecan increased with culture time. These results suggest that this in situ gel system is useful for regenerating cartilage in vitro and for minimally invasive therapy for cartilage defects.
Keywords: Alkali-treated collagen; Star polymer; Polyethylene glycol; Chondrocytes;

A previously reported “grafting and coating” method (J. Biomed. Mater. Res. (Appl. Biomater.) 63 (2002) 838) was modified and used to introduce stable collagen layer and incorporate basic fibroblast growth factor (bFGF) on PLLA scaffold surface to prepare tissue engineering scaffold with improved biocompatibility. To make the modification of the 3-D porous PLLA scaffold possible, grafting of polymethacrylic acid (PMAA) onto the PLLA surface was initiated by the –OOH/Fe2+ system instead of the UV light used in the former method. Water soluble carbodimmide chemistry was applied to graft collagen onto the PLLA scaffold surface, followed by physical coating of the collagen solution with or without basic fibroblast growth factor (bFGF). Surface modification of 2-D PLLA membrane was also done for fundamental understanding of the modification. The –COOH density on/in the PMAA grafted PLLA membrane/scaffold was measured by colorimetric method and the collagen content on/in the collagen immobilized PLLA membrane/scaffold was measured by ninhydrin method. Chondrocyte culturing on the collagen immobilized PLLA surfaces showed significantly improved cell spreading and growth. Incorporation of fibroblast growth factors in the collagen layer further enhanced the cell growth. This convenient and effective method can be used to prepare bioactive scaffolds with extra cellular matrix (ECM)-mimic composition for tissue engineering.
Keywords: PLLA; Surface modification; Scaffold; Tissue engineering; Growth factor;

Nanofiber alignment and direction of mechanical strain affect the ECM production of human ACL fibroblast by Chang Hun Lee; Ho Joon Shin; In Hee Cho; Young-Mi Kang; In Ae Kim; Ki-Dong Park; Jung-Woog Shin (1261-1270).
The effects of fiber alignment and direction of mechanical stimuli on the ECM generation of human ligament fibroblast (HLF) were assessed. The nanofiber matrix was fabricated using electrospinning technique. To align the nanofibers, a rotating target was used. The HLFs on the aligned nanofibers were spindle-shaped and oriented in the direction of the nanofibers. The degree of ECM production was evaluated by comparing the amount of collagen on aligned and randomly oriented structures. Significantly more collagen was synthesized on aligned nanofiber sheets, although the proliferation did not differ significantly. This suggests that the spindle-shape observable in intact ligaments is preferable in producing ECM. To evaluate the effect of strain direction on the ECM production, HLFs were seeded on parallel aligned, vertically aligned to the strain direction, and randomly oriented nanofiber sheets attached to Flexcell® plates. After a 48-h culture, 5% uniaxial strain was applied for 24 h at a frequency of 12 cycles/min. The amounts of collagen produced were measured 2 days after halting the strain application. The HLFs were more sensitive to strain in the longitudinal direction. In conclusion, the aligned nanofiber scaffold used in this study constitutes a promising base material for tissue-engineered ligament in that it provides more preferable biomimetic structure, along with proper mechanical environment.
Keywords: Ligament tissue engineering; Aligned nanofiber; Biomimetic material; Mechanical stimulation;

The role of macrophages in osteolysis of total joint replacement by Eileen Ingham; John Fisher (1271-1286).
The osteolysis associated with conventional polyethylene on metal total joint replacements is associated with the formation of an inflamed periprosthetic membrane rich in macrophages, cytokines and implant-derived wear particles. There is a wealth of evidence to indicate that the presence and activation of macrophages in the periprosthetic tissues around joint replacements is stimulated by UHMWPE particles. Particles within the size range 0.1–1.0 μm have been shown to be the most reactive. Animal studies have provided increasing evidence that, of the milieu of cytokines produced by particle-stimulated macrophages, TNF-α is a key cytokine involved in osteolysis. Recent advances in the understanding of the mechanisms of osteoclastogenesis and osteoclast activation at the cellular and molecular level have indicated that bone marrow-derived macrophages may play a dual role in osteolysis associated with total joint replacement. Firstly, as the major cell in host defence responding to UHMWPE particles via the production of cytokines and secondly as precursors for the osteoclasts responsible for the ensuing bone resorption.
Keywords: Macrophage; UHMWPE; Osteolysis; Osteoclast; Cytokines; Hip replacement;

Poly(N-isopropylacrylamide)-graft-polypropylene membranes containing adsorbed antibody for cell separation by Aiko Okamura; Midori Itayagoshi; Taeko Hagiwara; Manae Yamaguchi; Toshiyuki Kanamori; Toshio Shinbo; Pi-Chao Wang (1287-1292).
We developed a novel selective cell-separation method based on using a poly(N-isopropylacrylamide)-graft-polypropylene (PNIPAAm-g-PP) membrane containing adsorbed monoclonal antibody specific to the target cell. This membrane was prepared by plasma-induced polymerization and soaking in an antibody solution at 37°C.Poly(N-isopropylacrylamide) has a thermoresponsive phase transition: at 32°C water-insoluble (hydrophobic) and water-soluble (hydrophilic) states interconvert. Adsorption of antibody onto PNIPAAm-g-PP membrane at 37°C and its desorption at 4°C was verified by fluorescence-microscopy of the PNIPAAm-g-PP membrane after soaking it in fluorescein-conjugated goat anti-mouse IgG in phosphate-buffered saline. PNIPAAm-g-PP membranes containing adsorbed anti-mouse CD80 monoclonal antibody preferentially captured mouse-CD80 transfected cells at 37°C compared with membranes lacking antibody or containing anti-mouse CD86 monoclonal antibody. Detachment of captured cells from PNIPAAm-g-PP membranes was facilitated by washing at 4°C because of the thermoresponsive phase transition of PNIPAAm. With this method, mouse CD80- or mouse CD86-transfected cells were enriched from a 1:1 cell suspension to 72% or 66%, simply and with high yield.
Keywords: Poly(N-isopropylacrylamide)-graft-polypropylene; Plasma-induced graft polymerization; Monoclonal antibody; Cell separation; Membrane;

Ocular release of timolol from molecularly imprinted soft contact lenses by Haruyuki Hiratani; Akihito Fujiwara; Yuka Tamiya; Yuri Mizutani; Carmen Alvarez-Lorenzo (1293-1298).
The aim of this study was to evaluate “in vivo” the usefulness of molecular imprinting technology to obtain therapeutic soft contact lenses capable of prolonging the permanence of timolol in the precorneal area, compared to conventional contact lenses and eyedrops. Soft contact lenses (diameter 14 mm, center thickness 0.08 mm) consisted of N,N-diethylacrylamide (DEAA; main component of the matrix), methacrylic acid (MAA; functional monomer) and ethylene glycol dimethacrylate (EGDMA; cross-linker) were prepared by the conventional methodology (non-imprinted) or by applying a molecular imprinting technique using timolol as the template (imprinted ones). After washing and reloading, timolol release studies carried out in rabbits showed that the soft contact lenses made by the molecular imprinting method (34 μg dose) provided measurable timolol concentrations in the tear fluid for 2.0- and 3.0-fold longer than the non-imprinted contact lenses (21 μg dose) and eyedrops (doses of 34 and 125 μg), respectively. Furthermore, the area under the timolol concentration–time curve (AUC) was 3.3- and 8.7-fold greater for imprinted contact lenses than non-imprinted contact lenses and eyedrops, respectively. The timolol concentration of the eyedrops did not affect the precorneal residence time of drug significantly. On the other hand, timolol loading capacity of the contact lenses was improved by the molecular imprinting method; the sustaining of the drug levels in the tear fluid being proportional to the loading capacity of the contact lenses. These results indicate that imprinted soft contact lenses are promising drug devices able to provide greater and more sustained drug concentrations in tear fluid with lower doses than conventional eyedrops.
Keywords: Hydrogel; Molecular imprinting; Timolol; Contact lens; Controlled drug release;

Development and characterization of biodegradable nanospheres as delivery systems of anti-ischemic adenosine derivatives by Alessandro Dalpiaz; Eliana Leo; Federica Vitali; Barbara Pavan; Angelo Scatturin; Fabrizio Bortolotti; Stefano Manfredini; Elisa Durini; Flavio Forni; Barbara Brina; Maria Angela Vandelli (1299-1306).
We report a preliminary study concerning the encapsulation modalities in nanoparticles of the anti-ischemic drug N6-cyclopentyladenosine (CPA) and its pro-drug 5′-octanoyl-CPA (Oct-CPA). The release of these compounds and the related pro-drug stability effects in human whole blood have been tested. Moreover, the influence of the delivery systems on CPA interaction toward human adenosine A1 receptor has been analysed. The nanospheres were prepared by nanoprecipitation or double emulsion solvent evaporation method using poly(lactic acid) and recovered by gel filtration or ultracentrifugation or dialysis. Free and encapsulated Oct-CPA was incubated in fresh blood and its stability was analysed with HPLC. Quite spherical nanoparticles with mean diameters ranging between 210±50 and 390±90 nm were obtained. No encapsulation occurred when CPA was used. Satisfactory results concerning drug content (0.1–1.1% w/w) and encapsulation efficiency (6–56%) were achieved when Oct-CPA was employed. The controlled release of the pro-drug was achieved, being released within a range of 1–4 h, or very slowly, depending on nanoparticle preparations. The hydrolysis rate of Oct-CPA in human whole blood appeared stabilized in human whole blood with modalities related to the release patterns. The presence of all nanoparticle preparations did not interfere with CPA interaction at its action site.
Keywords: Nanoparticles; Controlled drug release; Polylactic acid; N6-Cyclopentyladenosine; Pro-drug; Stability;

Preparation and physicochemical characterization of biodegradable nerve guides containing the nerve growth agent sabeluzole by Geert Verreck; Iksoo Chun; Yufu Li; Ram Kataria; Qiang Zhang; Joel Rosenblatt; Annelies Decorte; Koen Heymans; Jef Adriaensen; Monique Bruining; Marie Van Remoortere; Herman Borghys; Theo Meert; Jef Peeters; Marcus E. Brewster (1307-1315).
The objective of this study was to develop and characterize a biodegradable drug-loaded nerve guide for peripheral nerve regeneration. Sabeluzole, a nerve growth agent, was selected as model compound. Four biodegradable polymers were selected for this study: a copolymer of polylactic acid and polycaprolactone (PCL); a copolymer of polyglycolic acid and polycaprolactone PCL; a copolymer of PCL/polydioxanone (PDO) and PDO. Placebo and drug loaded nerve guides were obtained by melt compression and melt extrusion.It was observed that melt compression and melt extrusion are feasible techniques to prepare the nerve guides. Based on the physicochemical characterization, all samples show absence of crystalline sabeluzole, indicating the formation of an amorphous dispersion. The in vitro release measurements show that the release of sabeluzole is complete, reproducible and can be controlled by the proper selection of the polymer. The release mechanism for all samples follows Fickian release behaviour.
Keywords: Sabeluzole; Nerve guide; Melt compression; Melt extrusion;

Rietveld refinements and spectroscopic studies of the structure of Ca-deficient apatite by Rory M. Wilson; James C. Elliott; Stephanie E.P. Dowker; Luis M. Rodriguez-Lorenzo (1317-1327).
Nine samples of Ca-deficient apatite (Ca-def Ap) were prepared from suspensions of CaHPO4 (monetite) at 90°C by raising the pH from ∼4 through release of NH3 produced by the hydrolysis of urea. Products were dried at 100°C for 24 h and studied by chemical analyses, X-ray powder diffraction (XRPD) (and Rietveld analysis of this data), Ca/P ratio determination (quantitative phase analysis of samples after heating to 900°C from Rietveld analysis of XRPD data), scanning electron microscopy, He pycknometry, 1H and 31P MAS NMR spectrometry and Fourier transform infrared and Raman spectroscopy. All samples contained apatite, but three also contained monetite. Infrared and Raman spectroscopy confirmed the presence of HPO4 2− and absence of carbonate ions in the six monetite-free samples. Mean results for the six samples were: a=9.4320(40), c=6.8751(31)  Å; unit cell formula from chemical analysis neglecting protonation of phosphate ion, Ca9.303(50)(PO4)6(OH)0.606(99).1.97(12)H2O; theoretical density 3.10 g cm−3; experimental density (mean for three samples) 3.15 g cm−3; and Ca/P mole ratio from chemical analysis and phase analysis after heating to 900°C, 1.550(8) and 1.550(2), respectively. An earlier assignment of a line at 6 ppm in the 1H NMR spectrum of similar samples to HPO4 2− ions could not be confirmed; hence no information about the HPO4 2− ion content could be derived, in disagreement with the previous NMR study. A shoulder at ∼0.9 ppm relative to 85 wt% H3PO4 in the 31P NMR spectrum was assigned to HPO4 2− ions. Occupancies from the Rietveld structure refinements indicated preferential loss of Ca from Ca2 sites compared with Ca1, but the loss was substantially smaller than expected from chemical analyses. It is suggested that imperfect modelling of the structure in the refinement, particularly disorder associated with the Ca2 site, resulted in errors in Ca2 occupancies. The P–O bonds were slightly shorter than those in stoichiometric hydroxyapatite, rather than longer as might be expected from protonation of phosphate tetrahedra. However, consideration of known acid phosphate structures indicated that it was unlikely that the increase in P–O lengths would be sufficient to be detected. The observed decrease was tentatively assigned to the presence of Ca2+ ion vacancies.
Keywords: Rietveld refinement; Apatite structure; MAS NMR; FTIR; Raman; X-ray diffraction;