Biomaterials (v.26, #25)

Calendar (I).

Systematic variation of interfacial phase reactivity in dental nanocomposites by Kristen S. Wilson; Kai Zhang; Joseph M. Antonucci (5095-5103).
This study was designed to determine the effect of varying the chemistry of the interfacial phase on critical composite properties in dental nanocomposite materials. Silica nanoparticles were silanized with varying ratios of 3-methacryloxypropyltrimethoxysilane (MPTMS) and octyltrimethoxysilane (OTMS) while keeping the total amount of silane constant at 10% by mass fraction relative to the mass of filler. The silanized nanoparticles were mixed into a dimethacrylate resin (60% filler by mass fraction). The mechanical properties of the uncured pastes were assessed by compression testing between parallel plates. The composites were photo-cured and tested by biaxial flexure and three-point bend flexure testing. Fracture surfaces were analyzed by field-emission scanning electron microscopy (FE-SEM). At maximized filler mass fractions, the workabilities of the uncured pastes were better maintained as the fraction of OTMS in the interphase increased relative to MPTMS. The flexure strengths and moduli of the MPTMS silanized and dual silanized composites were similar but decreased as OTMS mass fractions in the silane mixture increased to 7.5% and 10%. FE-SEM images revealed evidence for phase separation in the composites containing silica silanized with high fractions of OTMS. Among the potential practical benefits of dual silanized nanoparticles are the improved workability of composite pastes with higher filler loadings that should lead to higher modulus composites with lower polymerization shrinkage.
Keywords: Composite; Dental restorative material; Mechanical properties; Nanoparticle; Silane; Surface treatment;

Evidence of chemical bonding to hydroxyapatite by phosphoric acid esters by Baiping Fu; Xuemei Sun; Weixin Qian; Yanqing Shen; Ranran Chen; Matthias Hannig (5104-5110).
Phosphoric acid esters (PAEs) have been used as a self-etching primer for composite-to-enamel bonding in adhesive dentistry. However, the chemical mechanism of their interactions with hydroxyapatite (HA) is not clear. In the present study, HA particles were mixed with Resulcin AquaPrime (Merz Co.) priming agent that contains a mixture of PAEs, and dried. The primer, HA and the mixture of both were analyzed by FTIR. Primed and untreated HA discs were analyzed with attenuated total reflectance (ATR). After AquaPrime+D2O (1:1) was mixed with HA or Ca(OD)2 for 48 h, the primer and both mixtures were analyzed with 31P NMR in D2O. The solid and the liquid separated from both mixtures were analyzed with 31P NMR in CDCl3. The primer's characteristic bands (νC=O, νC=C) were found on the primer-subtracted mixture's spectrum and the primed HA disc. 31P NMR data indicated that the reactions of PAEs with HA produced PAEs–HA complexes, and were not a simple acid–base reaction like those with Ca(OD)2, either in liquid or in solid. It is concluded that phosphoric acid esters can decalcify and adhere to hydroxyapatite simultaneously.
Keywords: Dental adhesive; Adhesion mechanism; FTIR; Hydroxyapatite;

Surface characterization of silver-doped bioactive glass by E. Vernè; S. Di Nunzio; M. Bosetti; P. Appendino; C. Vitale Brovarone; G. Maina; M. Cannas (5111-5119).
A bioactive glass belonging to the system SiO2-CaO-Na2O was doped with silver ions by ion exchange in molten salts as well as in aqueous solution. The ion exchange in the solution was done to check if it is possible to prepare an antimicrobial material using a low silver content. The doped glass was characterized by means of X-ray diffraction, SEM observation, EDS analysis, bioactivity test (soaking in a simulated body fluid), leaching test (GFAAS analyses) and cytotoxicity test. It is demonstrated that these surface silver-doped glasses maintain, or even improve, the bioactivity of the starting glass. The measured quantity of released silver into simulated body fluid compares those reported in literature for the antibacterial activity and the non-cytotoxic effect of silver. Cytotoxicity tests were carried out to understand the effect of the doped surfaces on osteogenic cell adhesion and proliferation.
Keywords: Silver; Antibacterial; Bioactive glasses; Cytotoxicity;

Titanium has been a successful implant material owing to its excellent strength to weight ratio, toughness, and bioinert oxide surface. Significant progress has been made on the improvement of titanium's bioactivity by coating its oxide surface with calcium phosphates and bioactive molecules. Here, we report on the coating of titanium with a poly(l-lysine)-calcium phosphate hybrid material with a nanoscale texture. This hybrid coating was grown by first nucleating seed crystals of calcium phosphate, directly on the Ti surface and then exposing this surface to solutions containing Ca2+, PO4 3−, and poly(l-lysine). The resultant hybrid coating was characterized by electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, and elemental analysis. This material contained 14% by weight poly(l-lysine), and this organic component decreased greatly the dimensions of the surface features, thus enhancing surface area relative to the inorganic control. The highly textured hybrid material was more susceptible than the control to acidic and enzymatic degradation. The amino acid cysteine was covalently linked to the hybrid material, demonstrating the potential of this coating for further functionalization. These hybrid coatings may prove useful in enhancing the bioactivity of titanium.
Keywords: Titanium; Calcium phosphate; Poly(l-lysine); Hybrid; Degradation; Covalent attachment;

TEGDMA induces mitochondrial damage and oxidative stress in human gingival fibroblasts by Mathieu Lefeuvre; Wafaa Amjaad; Michel Goldberg; Lena Stanislawski (5130-5137).
Free monomers including triethylene glycol dimethacrylate (TEGDMA) are released by resin composite. Recent studies in vitro have demonstrated that TEGDMA induced GSH depletion and production of radical oxygen species (ROS) in human gingival fibroblasts (HGF) but the exact mechanism of these events remains unclear. Our purpose is to investigate the origin of ROS production. TEGDMA induces a rapid (within 30 min) and drastic depletion of ATP concomitant with the GSH depletion. After 3 h incubation, TEGDMA induced an increase of lipid peroxidation associated with LDH leakage. Our data also showed that TEGDMA produced damage at mitochondrial level. This is demonstrated by the collapse of mitochondrial membrane potential (MMP) in HGF treated with TEGDMA. The protective effect of carbonylcyanide m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation on lipid peroxidation and LDH leakage suggests that mitochondria can be implicated in these events. Trolox, a soluble derivative of Tocopherol, weakly prevents ATP but not GSH depletion and totally protects the cells against lipid peroxidation, MMP collapse and cell death. Thus, the present results suggest that TEGDMA induces lipid peroxidation and mitochondrial damage, which contribute to cell death.
Keywords: Fibroblasts; TEGDMA; Trolox; Lipid peroxydation; Glutathione; MMP;

The response of normal human osteoblasts to anionic polysaccharide polyelectrolyte complexes by Misao Nagahata; Ryusuke Nakaoka; Akira Teramoto; Koji Abe; Toshie Tsuchiya (5138-5144).
Polyelectrolyte complexes (PEC) were prepared from chitosan as the polycation and several synthesized functional anion polysaccharides, and their effects on cell attachment, morphology, proliferation and differentiation were estimated using normal human osteoblasts (NHOst). After a 1-week incubation, PEC made from polysaccharides having carboxyl groups as polyanions showed low viability of NHOst on it although the NHOst on it showed an enhancement in their differentiation level. On the other hand, NHOst on PEC made from sulfated or phosphated polysaccharides showed similar attachment and morphology to those on the collagen-coated dish. When the number of NHOst was estimated after 1 week, the number on the PEC was ranged from 70% to 130% of those on the collagen-coated dish, indicating few effects of these PEC on cell proliferation. In addition, NHOst on PEC films made from sulfated polysaccharides differentiated to a level very similar to that observed on the collagen-coated dish, indicating that these PEC films maintain the normal potential of NHOst to both proliferate and differentiate. Measurement of gap junctional intercellular communication of NHOst on PEC revealed that PEC did not inhibit communication, suggesting that PEC films have few effects on cell homeostasis. Thus, PEC made from the sulfated polysaccharide may be a useful material as a new scaffold for bone regeneration.
Keywords: Polyelectrolyte complex; Normal human osteoblasts; Cell proliferation; Cell differentiation; Gap junctional intercellular communication;

Repair of an intercalated long bone defect with a synthetic biodegradable bone-inducing implant by Masahiro Yoneda; Hidetomi Terai; Yuuki Imai; Takao Okada; Kazutoshi Nozaki; Hikaru Inoue; Shimpei Miyamoto; Kunio Takaoka (5145-5152).
Recombinant human bone morphogenetic protein (rhBMP)-2 in a block copolymer composed of poly-d,l-lactic acid with randomly inserted p-dioxanone and polyethylene glycol (PLA-DX-PEG) as a carrier and porous β-tricalcium phosphate (β-TCP) blocks were used to generate a new fully absorbable osteogenic biomaterial. The bone regenerability of the rhBMP-2/PLA-DX-PEG/β-TCP composite was studied in a critical-sized rabbit bone defect model. In an initial study, a composite of PLA-DX-PEG (250 mg) and β-TCP (300 mg) loaded with or without rhBMP2 (50 μg) was implanted into a 1.5 cm intercalated bone defect created in a rabbit femur. Defects were assessed by biweekly radiography until 8 weeks postoperatively. The bony union of the defect was recognized only in the BMP-loaded group. To obtain further data on biomechanical and remodeling properties, another BMP-loaded composites group was made and observed up to 24 weeks. All defects were completely repaired without residual traces of implants. Anatomical and mechanical properties of the repaired bone examined by histology, 3-dimensional CT (3D-CT) and mechanical testing were essentially equivalent to the nonoperated-on femur at 24 weeks. These experimental results indicate that fully absorbable rhBMP-2/PLA-DX-PEG/β-TCP is a promising composite having osteogenicity efficient enough for repairing large bone defects.
Keywords: BMP (bone morphogenetic protein); Bone tissue engineering; Biodegradation; Osteoconduction; Drug delivery; Calcium phosphate;

The surface of titanium implants is in direct contact with host tissue and plays a critical role in determining biocompatibility. Fibronectin (FN) and vitronectin (VN) are major cell adhesive proteins found in the extracellular matrix (ECM) of various tissues, and in circulating blood. The aim of this study was to evaluate the engineered biomimetic surface of titanium by using recombinant fragment of FN8–10 and VNNTD that contains the binding site for integrins. MC3T3-E1 cells seeded upon the FN8−10-coated titanium showed a marked increase in cell adhesion, proliferation, and differentiation over VNNTD-coated titanium. In addition, we confirmed that the surface properties of titanium prefer for FN8–10 over VNNTD (p<0.05) in protein adhesion. These results suggest that the FN8–10-modified titanium surface can be used to improve the osseointegration of titanium implants by enhancing bone formation.
Keywords: Titanium; Fibronectin; Protein adsorption; Dental implants; Biomimetic material;

Multilineage differentiation of human mesenchymal stem cells in a three-dimensional nanofibrous scaffold by Wan-Ju Li; Richard Tuli; Xiaoxue Huang; Patrice Laquerriere; Rocky S. Tuan (5158-5166).
Functional engineering of musculoskeletal tissues generally involves the use of differentiated or progenitor cells seeded with specific growth factors in biomaterial scaffolds. Ideally, the scaffold should be a functional and structural biomimetic of the native extracellular matrix and support multiple tissue morphogenesis. We have previously shown that electrospun, three-dimensional nanofibrous scaffolds that morphologically resemble collagen fibrils are capable of promoting favorable biological responses from seeded cells, indicative of their potential application for tissue engineering. In this study, we tested a three-dimensional nanofibrous scaffold fabricated from poly( ε -caprolactone) (PCL) for its ability to support and maintain multilineage differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs) in vitro. hMSCs were seeded onto pre-fabricated nanofibrous scaffolds, and were induced to differentiate along adipogenic, chondrogenic, or osteogenic lineages by culturing in specific differentiation media. Histological and scanning electron microscopy observations, gene expression analysis, and immunohistochemical detection of lineage-specific marker molecules confirmed the formation of three-dimensional constructs containing cells differentiated into the specified cell types. These results suggest that the PCL-based nanofibrous scaffold is a promising candidate scaffold for cell-based, multiphasic tissue engineering.
Keywords: Electrospinning; Nanofiber; Mesenchymal stem cell; Adipogenesis; Chondrogenesis; Osteogenesis;

Temperature-responsive culture dishes immobilized with insulin have been fabricated and studied to shorten cell culture periods by facilitating more rapid cell proliferation. Cells are recovered as contiguous cell sheets simply by temperature changes. Functionalized culture dishes were prepared by previously reported electron beam grafting copolymerization of N-isopropylacrylamide (IPAAm) with its carboxylate-derivatized analog, 2-carboxyisopropylacrylamide (CIPAAm), having similar molecular structure to IPAAm but with carboxylate side chains to tissue culture polystyrene dishes. Insulin was then immobilized onto culture dishes through standard amide bond formation with CIPAAm carboxylate groups. Adhesion and proliferation of bovine carotid artery endothelial cells (ECs) were examined on these insulin-immobilized dishes. Insulin immobilization was shown to promote cell proliferation in serum-supplemented medium. Increasing the grafted CIPAAm content on the tissue culture surfaces reduces cell adhesion and proliferation, even though these surfaces contained increased amounts of immobilized insulin. This result implies that a discrete balance exists between the amount of CIPAAm-free carboxylate groups and immobilized insulin for optimum cell proliferative stimulation. Cells grown on the insulin-immobilized surfaces can be recovered as contiguous cell monolayers simply by lowering culture temperature, without need for exogenous enzyme or calcium chelator additions. In conclusion, insulin-modified thermoresponsive culture dishes may prove useful for advanced cell culture and tissue engineering applications since they facilitate cell proliferation, and cultured cells can be recovered as viable contiguous monolayers by merely reducing culture temperature.
Keywords: Poly(N-isopropylacrylamide) (PIPAAm); 2-Carboxyisopropylacrylamide (CIPAAm); Insulin; Thermoresponsive surfaces; Cell proliferation; Cell sheet; Tissue engineering;

Cytocompatibility of self-assembled β-hairpin peptide hydrogel surfaces by Juliana K. Kretsinger; Lisa A. Haines; Bulent Ozbas; Darrin J. Pochan; Joel P. Schneider (5177-5186).
MAX1 is a 20 amino acid peptide that undergoes triggered self-assembly to form a rigid hydrogel. When dissolved in aqueous solutions, this peptide exists in an ensemble of random coil conformations rendering it fully soluble. The addition of an exogenous stimulus results in peptide folding into β-hairpin conformation. This folded structure undergoes rapid assembly into a highly crosslinked hydrogel network. DMEM cell culture media is one stimulus able to initiate folding and consequent self-assembly of MAX1. The cytocompatibility of this gel towards NIH 3T3 murine fibroblasts is demonstrated. Gels were shown to be non-toxic to the fibroblast cells. MAX1 hydrogels also foster the ability of the cells to attach to the hydrogel scaffold in the absence or presence of serum proteins. Additionally MAX1 hydrogels were able to support fibroblast proliferation to confluency with little effect on the rheological properties of the scaffold. MAX1 hydrogels meet the preliminary mechanical and cytocompatibiltiy requirements of a tissue engineering scaffold.
Keywords: Cell adhesion; Cell proliferation; Cell viability; Fibroblast; Hydrogel; Peptide;

Tissue engineered cartilage on collagen and PHBV matrices by Gamze Torun Köse; Feza Korkusuz; Aykut Özkul; Yasemin Soysal; Taner Özdemir; Cemil Yildiz; Vasif Hasirci (5187-5197).
Cartilage engineering is a very novel approach to tissue repair through use of implants. Matrices of collagen containing calcium phosphate (CaP–Gelfix®), and matrices of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) were produced to create a cartilage via tissue engineering. The matrices were characterized by scanning electron microscopy (SEM) and electron diffraction spectroscopy (EDS). Porosity and void volume analysis were carried out to characterize the matrices. Chondrocytes were isolated from the proximal humerus of 22 week-old male, adult, local albino rabbits. For cell type characterization, Type II collagen was measured by Western Blot analysis. The foams were seeded with 1×106 chondrocytes and histological examinations were carried out to assess cell–matrix interaction. Macroscopic examination showed that PHBV (with or without chondrocytes) maintained its integrity for 21 days, while CaP–Gelfix® was deformed and degraded within 15 days.Cell-containing and cell-free matrices were implanted into full thickness cartilage defects (4.5 mm in diameter and 4 mm in depth) at the patellar groove on the right and left knees of eight rabbits, respectively. In vivo results at 8 and 20 weeks with chondrocyte seeded PHBV matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body reaction. In CaP–Gelfix® matrices, fibrocartilage formation and bone invasion was noted in 20 weeks. Cells maintained their phenotype in both matrices. PHBV had better healing response than CaP–Gelfix®.Both matrices were effective in cartilage regeneration. These matrices have great potential for use in the repair of joint cartilage defects.
Keywords: Cartilage; Tissue engineering; Collagen; PHBV; Biomaterials;

The objective of this study was to investigate the effect of combining two biomaterials on osteoblast proliferation, differentiation and mineralised matrix formation in vitro. The first biomaterial has a well-defined architecture and is known as PolyHIPE polymer (PHP). The second biomaterial is a biologically inspired self-assembling peptide hydrogel (RAD16-I, also called PuraMatrix™) that produces a nanoscale environment similar to native extracellular matrix (ECM). Our work investigates the effect of combining RAD16-I with two types of PHP (HA (Hydroxyapatite)-PHP and H (Hydrophobic)-PHP) and evaluates effects on osteoblast growth and differentiation. Results demonstrated successful incorporation of RAD16-I into both types of PHP. Osteoblasts were observed to form multicellular layers on the combined biomaterial surface and also within the scaffold. Dynamic cell seeding and culturing techniques were compared to static seeding methods and produced a more even distribution of cells throughout the constructs. Cells were found to penetrate the scaffold to a maximum depth of 3 mm after 35 days in culture. There was a significant increase in cell number in H-PHP constructs coated with RAD16-I compared to H-PHP alone. Our results show that RAD16-I enhances osteoblast differentiation and indicates that the incorporation of this peptide provides a more permissive environment for osteoblast growth. We have developed a microcellular polymer containing a nanoscale environment to enhance cell: biomaterial interactions and promote osteoblast growth in vitro.
Keywords: Bone tissue engineering; Cell adhesion; Hydrogel; Osteoblast; Osteogenesis; Adhesion mechanism;

Poly(ethylene glycol) hydrogels were synthesized with pendant peptide functionalities to examine the influence of synergistic peptide sequences on osteoblast adhesion, spreading, and function. Specifically, acrylated monomers were prepared that contained the peptide sequence, Arg–Gly Asp (RGD), as well as monomers with RGD plus its synergy site, Pro–His–Ser–Arg–Asn (PHSRN), linked via a polyglycine sequence to recapitulate the native spacing of fibronectin. The colocalized RGD–PHSRN sequence improved osteoblast adhesion, spreading, and focal contact formation when compared to RGD alone. In addition, proliferation, metabolic activity, and levels of alkaline phosphatase production, a common marker for osteoblast function, were statistically higher for the colocalized peptide sequences at 1 day, 1 week, and 2 weeks, when compared to control surfaces. Interestingly, increases were not observed in all areas of cell function, as extracellular matrix (ECM) production was the lowest on gels functionalized with the colocalized peptide sequence. This result was attributed to strong receptor–ligand interactions initiating signal transduction cascades that down-regulate ECM production.
Keywords: Hydrogels; Poly(ethylene) glycol; RGD peptide; Extracellular matrix; Bone tissue engineering;

Collagen-derived gelatin/hydroxyapatite (HA) nanocomposites were biomimetically synthesized for hard tissue engineering scaffold. In vitro osteoblastic cellular responses to the nanocomposites were assessed in comparison with those conventionally mixed gelatin–HA composites. A three-dimensional culture method involving floating cells in a culture medium was introduced to assist in the initial attachment of the cells to the scaffolds, and the proliferation and differentiation behaviors of the cells were examined. The osteoblastic MG63 cells attached to the nanocomposites to a significantly higher degree and subsequently proliferated more. The alkaline phosphatase (ALP) activity and osteocalcin produced by the cells were significantly higher on the nanocomposite scaffolds than on the conventional composite scaffolds. These improved cellular responses on the nanocomposites are considered to result from the increased ionic release and serum protein adsorption on the nanocomposites, which was derived from the different structural and morphological characteristics, i.e., the nanocomposite scaffolds retained less-crystallized and smaller-sized apatite crystals and a more well-developed pore configuration than the conventional ones. Based on these findings, the biomimetically synthesized nanocomposite scaffolds are believed to be potentially useful in hard tissue regeneration and tissue engineering fields.
Keywords: Nanocomposites; Hydroxyapatite (HA)–Gelatin; Tissue engineering scaffold; Osteoblast response;

Bone tissue engineering on amorphous carbonated apatite and crystalline octacalcium phosphate-coated titanium discs by Robert J. Dekker; Joost D. de Bruijn; Martin Stigter; Florence Barrere; Pierre Layrolle; Clemens A. van Blitterswijk (5231-5239).
Poor fixation of bone replacement implants, e.g. the artificial hip, in implantation sites with inferior bone quality and quantity may be overcome by the use of implants coated with a cultured living bone equivalent. In this study, we tested, respectively, amorphous carbonated apatite (CA)- and crystalline octacalcium phosphate (OCP)-coated discs for their use in bone tissue engineering. Subcultured rat bone marrow cells were seeded on the substrates and after 7 days of culture, the implants were subcutaneously implanted in nude mice for 4 weeks. After 7 days of culture, the cells had formed a continuous multi-layer that covered the entire surface of the substrates. The amount of cells was visually higher on the crystalline OCP-coated discs compared to the amorphous CA-coated discs. Furthermore, the amorphous CA-coated discs exhibited a visually higher amount of mineralized extracellular matrix compared to the crystalline OCP-coated discs. After 4 weeks of implantation, clear de novo bone formation was observed on all discs with cultured cells. The newly formed bone on the crystalline OCP-coated discs was more organized and revealed a significantly higher volume compared to the amorphous CA-coated discs. The percentage of bone contact with the discs was also significantly higher on the OCP-coated discs. Overall, the results suggest that a crystalline OCP coating is more suitable for bone tissue engineering than an amorphous CA coating.
Keywords: Bone tissue engineering; Mesenchymal stem cells; Calcium phosphate coating;

Thrombospondin 1 as possible key factor in the hemocompatibility of endocoronary prostheses by Jan Hoffmann; Perikles Simon; Anja K. Zimmermann; Michael Lemancyk; Thomas Walter; Martin Beyer; Hans-Martin Hoffmeister; Gerhard Ziemer; Hans P. Wendel (5240-5250).
Intracoronary stenting has markedly improved the patency of native coronary arteries after percutaneous transluminal coronary angioplasty (PTCA). Advances in stent technology and design, including drug releasing stents, have contributed to reduce the long-term restenosis rate. However, stenosis caused by neointimal hyperplasia, vascular remodeling and thrombosis is still a major problem after endocoronary stent procedures. This study focuses on differential gene expression of circulating peripheral blood cells after 90 min exposure to stents to search for initially activated cellular pathways, which may foster restenosis. Fresh human whole blood (1 IU heparin/ml), taken from non-medicated healthy volunteers, was incubated under flow conditions in an in vitro closed-loop stent-testing model (modified Chandler-Loop). Differential gene expression compared to resting conditions and to the experimental controls was investigated by a DNA-microarray technique encoding for over 17,000 genes simultaneously. As expected, a large variety of genes showed differential gene expression. Interestingly, Thrombospondin 1 (TSP-1), which plays a key role in initial immune defense, was found to be the most markedly up-regulated gene. We propose TSP-1 expression as an early indicator for the activation of immune responses following intracoronary stenting. After clarifying the participation of TSP-1 in vivo, future studies will therefore focus on TSP-1 as a potential prognostic factor, which may also help to develop and control new surface materials with an improved biocompatibility.
Keywords: Stent; Biocompatibility; Thrombospondin 1; DNA-microarray;

Effectiveness of hydroxyapatite-vancomycin bone cement in the treatment of Staphylococcus aureus induced chronic osteomyelitis by Uwe Joosten; Alexander Joist; G. Gosheger; Ulf Liljenqvist; Burkhard Brandt; Christof von Eiff (5251-5258).
In the field of local application of antimicrobials, a number of novel drugs and/or new drug delivery systems have been developed in recent years. The present study aimed to investigate hydroxyapatite cement (HAC) as a carrier for vancomycin in the treatment of chronic osteomyelitis due to Staphylococcus aureus strains with various mechanisms of resistance. The release of vancomycin from standard test cylinders was determined in vitro and the efficacy of the delivery system was measured in vivo using a rabbit model of chronic osteomyelitis.First, powdered HAC was mixed with vancomycin at 80, 160 and 240 mg/g. After hardening, formed cylinders were eluted in phosphate buffer and antibiotic release was measured by agar diffusion. High levels of release (1512±318 to 1937±336 μg/ml) were obtained for 12 to 20 days depending on the dosage of vancomycin.Additionally, bone infection was induced in the tibia of 30 New Zealand white rabbits by injecting either a methicillin-resistant S. aureus strain (MRSA) or a S. aureus strain with a small colony variant (SCV) phenotype. After 3 weeks (chronic infection), all animals were treated by debridement. Moreover, group 1 (challenged with SCVs) and group 2 (challenged with MRSA) were treated by filling the marrow with HAC alone, whereas in groups 3 (SCVs) and 4 (MRSA) the marrow was filled with HAC/vancomycin (160 mg/g). After 6 weeks all animals were sacrificed.At 3 weeks, pathogens were detected in 24 of 30 animals. All swabs of the control groups, positive for S. aureus on day 21, were also positive on day 42 and S. aureus strains recovered were shown to be clonal to the strains used for induction of osteomyelitis. By contrast, no growth was found in the treatment group following 7 days of incubation in BHI bouillon. HAC/vancomycin-treated animals showed no histological evidence of infection on day 42. In the other groups, different stages of chronic osteomyelitis were found histologically. No local or systemic side effects due to HAC or vancomycin were seen.HAC is an effective carrier material for antibiotic compounds even in refractory infections due to MRSA or S. aureus SCVs.
Keywords: Chronic osteomyelitis; Rabbit model; Hydroxyapatite bone cement; Vancomycin; Local application; Staphylococcus aureus; Methicillin-resistant S. aureus (MRSA); Small colony variants (SCVs);

Poly(ethylene glycol)s modified with fluorocarbon end groups are capable of in situ transition from an injectable liquid to a viscoelastic hydrogel by hydrophobic interaction of the end groups; this class of materials is useful for a variety of biomedical applications, including sustained protein release. The hydrogel state can be transformed into an injectable state by the addition of a toxicologically acceptable organic solvent, such as N-methyl pyrrolidone; after injection, this solution quickly returns to a gel state by diffusion of the water-miscible organic solvent into the surrounding environment. In vitro characterization of sustained release of human growth hormone (hGH) using this injectable depot shows that hGH remains stable inside the hydrogel formed, and demonstrates more than 2 weeks of prolonged release of hGH complexed with Zn2+ ions without protein aggregation or initial burst.
Keywords: In situ forming; Sustained release; Human growth hormone; Diffusion;