Cathepsin K/TRAP: Can they be used to induce osteogenesis?

Most of the strategies developed in bone tissue engineering in the past three decades have been aimed to repair/regenerate the tissue with forming elements such as osteoblasts and bone morphogenetic proteins. All these materials are selected as they are known to induce bone formation. Since it is known that bone turnover in basic multicellular units (BMUs) is at equilibrium, inducing an imbalance in this process via molecules known to resorb and transmit resorption signals and therefore initiate activation in bone forming cells from adjacent tissue may offer a radical approach to bone regeneration. Possible targets for such an approach may include resorbing molecules such as tartrate resistant acid phosphatase (TRAP) and cathepsin K. Delivering these enzymes (TRAP and cathepsin K) and/or other molecules involved in bone resorption into bone defects and thus obtaining a concentration difference in the levels of these materials may induce bone forming cells to balance bone turnover, therefore inducing bone regeneration.

Separation of mesenchymal stem cells with magnetic nanosorbents carrying CD105 and CD73 antibodies in flow-through and batch systems.

The aim of this study is to develop magnetically loaded nanosorbents carrying specific monoclonal antibodies (namely CD105 and CD73) for separation of mesenchymal stem cells from cell suspensions. Super-paramagnetic magnetite (Fe3O4) nanoparticles were produced and then coated with a polymer layer containing carboxylic acid functional groups (average diameter: 153 nm and polydispersity index: 0.229). In order to obtain the nanosorbents, the monoclonal antibodies were immobilized via these functional groups with quite high coupling efficiencies up to 80%. These nanosorbents and also a commercially available one (i.e., microbeads carrying CD105 antibodies from Miltenyi Biotec., Germany) were used for separation of CD105+ and CD73+ mesenchymal stem cells from model cell suspension composed of peripheral blood (97.6%), human bone marrow cells (1.2%) and fibroblastic cells (1.2%). The initial concentrations of the CD105+ and CD73+ cells in this suspension were measured as 5.86% and 6.56%, respectively. A flow-through separation system and a very simple homemade batch separator unit were used. We were able to increase the concentration of CD105+ cells up to about 86% in the flow-through separation system with the nanosorbents produced in this study, which was even significantly better than the commercial one. The separation efficiencies were also very high, especially for the CD73+ cells (reached to about 64%) with the very simple and inexpensive homemade batch unit.

In vivo performance of antibiotic embedded electrospun PCL membranes for prevention of abdominal adhesions.

The aim of this study was to prepare nonwoven materials from poly(epsilon-caprolactone) (PCL) and their antibiotic containing forms by electrospinning, so as to prevent postsurgery induced abdominal adhesions in rats. epsilon-Caprolactone was first polymerized by ring-opening polymerization, and then it was processed into matrices composed of nanofibers by electrospinning. A model antibiotic (Biteral) was embedded within a group of PCL membranes. In the rat model, defects on the abdominal walls in the peritoneum were made to induce adhesion. The plain or antibiotic embedded PCL membranes were implanted on the right side of the abdominal wall. No membrane implantation was made on the left side of the abdominal wall that served as control. Macroscopical and histological evaluations showed that using these barriers reduces the extent, type, and tenacity of adhesion. The antibiotic embedded membranes significantly eliminated postsurgery abdominal adhesions, and also improved healing.

Attachment and growth of fibroblasts on poly(L-lactide/epsilon-caprolactone) scaffolds prepared in supercritical CO2 and modified by polyethylene imine grafting with ethylene diamine-plasma in a glow-discharge apparatus.

In this study, a copolymer of L-lactide and epsilon-caprolactone (Mn: 73,523, Mw: 127,990 and PI: 1.74) was synthesized by ring-opening polymerization by using stannous octoate as the catalyst. FTIR, 1H-NMR and DSC confirmed the copolymer formation. The copolymer films were prepared and a novel method was developed to produce highly porous sponges for potential use in tissue engineering. Films were subjected to supercritical CO2 at 3300 psi and 70 degrees C to create porous structures for production of possible tissue engineering scaffolds. The pore sizes were in the range of 40-80 microm. The copolymer films were pre-wetted with polyethylene imine (PEI) and then treated with ethylene diamine (EDA)-plasma in glow-discharge apparatus. Gas plasma surface modification of three-dimensional scaffolds fabricated by supercritical carbon dioxide technique was demonstrated to enhance cell adhesion, proliferation, and differentiation over 6 days in culture using L929 fibroblast cell line. Alkaline phosphatase (ALP) activity and glucose uptake in cell culture medium were followed in the cell culture experiments. Fibroblastic cell attachment and growth on the EDA-plasma treated scaffolds were rather low. However, both cell attachment and growth were significantly increased by PEI pre-treatment before EDA-plasma. The changes in ALP activity and glucose uptake also supported the cell growth behavior on these PEI and EDA-plasma treated scaffolds.

Synthesis and characterization of poly(glycerol-co-sebacate-co-ε-caprolactone) elastomers.

In this study, poly(glycerol-co-sebacate-co-ε-caprolactone) (PGSCL) elastomers were synthesized for the first time from the respective monomers. The structural analysis of PGSCL elastomers by nuclear magnetic resonance ((1)H-NMR) and Fourier transform infrared spectroscopy (FTIR) revealed that the elastomers have a high number of hydrogen bonds and crosslinks. X-ray diffraction (XRD) and thermal analysis indicated an amorphous state. Differential scanning calorimetry (DSC) analysis showed that the elastomers has a glass transition temperature (T(g)) of -36.96°C. The Young's modulus and compression strength values were calculated as 46.08 MPa and 3.192 MPa, respectively. Calculations based on acid number and end groups analysis revealed a number average molecular weight of 148.15 kDa. Even though the foaming studies conducted by using supercritical CO2 resulted in a porous structure; the obtained morphology tended to disappear after 48 h, leaving small cracks on the surface. This phenomenon was interpreted as an indication of self-healing due to the high number of hydrogen bonds. The PGSCL elastomers synthesized in this study are flexible, robust to compression forces and have self-healing capacity. Thanks to good biocompatibility and poor cell-adhesion properties, the elastomers may find diverse applications where a postoperative adhesion barrier is required.

Subcutaneous polymeric matrix system poly(HEMA-BGA) for controlled release of an anticancer drug (5-fluorouracil). I. Synthesis and structure.

The present study was an attempt to design a new polymer-drug composite system for local chemotherapy. Poly(hydroxyethylmethacrylate-bisglycolacrylate) carriers containing an anticancer drug, 5-fluorouracil, were prepared by low temperature radiation polymerization technique. By changing the relative amounts and the types of ingredients, drug loading and radiation dosage, polymer-drug composites with different structural properties were obtained. This paper presents the preparation procedure and analyses some of the structural properties of these novel composites.

Subcutaneous polymeric matrix system p(HEMA-BGA) for controlled release of an anticancer drug (5-fluorouracil). II: Release kinetics.

A subcutaneous polymeric drug delivery system, which consists of a polymeric matrix of poly(hydroxyethyl methacrylate-bisglycol acrylate), was developed. 5-fluorouracil was used as the model anticancer drug. Polymer-drug beads with a diameter of 3 mm were prepared by low-temperature radiation polymerization. In order to modify the release rate, polymeric beads with different composition, drug loading and crosslinking density were obtained. The kinetics of drug release were described by the expression Mt/M infinity = ktn. The diffusional release exponent 'n', which was calculated from the release curves, indicated that the mechanism of drug release from the polymeric matrix is due to the anomalous (non-Fickian) type of diffusion.

Poly(D,L-lactide/epsilon-caprolactone)/hydroxyapatite composites.

In this study, elastomeric D,L-lactide and epsilon-caprolactone copolymers with two different molecular weights (Mn: 108.000 and 40.000) were synthesized by ring-opening polymerization of the respective dimers by using stannous octoate as the catalyst, as a potential bone-filling material. The final ratio of D,L-lactide to epsilon-caprolactone obtained by 1NMR was 60/40 (comparing to the initial ratio of 50/50). Both copolymers were amorphous having Tg at around -21 degrees C. Different amounts of hydroxyapatite (HA) powder were loaded within the copolymers. These composites were easily shaped by hand. Mechanical properties of the composites changed with the HA loading and the molecular weight of the copolymer. The percent elongation decreased, while both the Young's modulus and yield point (stress) increased with the HA content. The copolymers were degraded within the Ringer solutions in about 6 weeks. The molecular weight distribution became broader during degradation. Incorporation of HA reduced the degradation rate.

In vivo degradation and release kinetics of chloramphenicol-loaded poly(D,L)-lactide sponges.

Poly(d,l)-lactide (PDLLA) homopolymer, with an average molecular weight of 20,000 daltons, was produced by the ring-opening polymerization of d,l-lactide in the presence of SnCl(2).2H(2)O as the catalyst. The PDLLA sponges loaded with chloramphenicol were prepared by a solvent evaporation technique. The drug loadings achieved were 14.84 and 25.23 mg for the PDLLA sponges with 35 and 70 mg total weights, respectively. These sponges were implanted in Wistar rats, and in vivo degradation, drug release, and tissue reactions were followed. The PDLLA sponges carrying no drug degraded with time linearly. Almost 80% of the sponges were degraded in about 180 days. While the drug-loaded PDLLA sponges were degraded much faster in 4 weeks (about 35% of the matrix was degraded), then the degradation slowed down significantly. Drug release from the sponges was parallel to the degradation. Almost 60% of the loaded drug released in 4 weeks. There were no acute inflammatory reactions in the initial period, either for the plain or for the drug-loaded PDLLA sponges. Macrophages and multinuclear giant cells start to appear after 7 days of implantation. The fibroblastic activity also started after the same period. After that, there were decreases in the number of some cells (neutrophils, lymphocytes, and macrophages), while multinuclear giant cells and fibroblastic activities gradually increased. Granulation tissue started at about 1 month, and new connective tissue was gradually formed until 180 days of implantation. There were significant numbers of inflammatory cells after 60 days, which were replaced by fibroblasts after 180 days. There was almost no significant neovascularization after 180 days, but implant fragmentation gradually increased (which slows the degradation) with time. It was concluded that this novel drug release sponge may be safely and effectively used as an active soft tissue-filling material.

Hepatocyte immobilization on PHEMA microcarriers and its biologically modified forms.

Polyhydroxyethylmethacrylate (PHEMA) based microcarriers with different bulk structures were prepared by a phase inversion polymerization technique. PHEMA surfaces were further modified chemically by glow-discharge treatment, and biologically by covalent attachment of fibrinogen and collagen. Hepatocytes were isolated from young male Wistar rats using an in situ portal vein collagenase perfusion technique. Freshly isolated hepatocytes were seeded at 6 x 10(5) cells/mL and microcarrier concentration was 10 g/L. Stationary microcarrier cultures were carried out in standard (nontissue culture) polystyrene petri dishes in a humidified 5% CO2 incubator at 37 +/- 0.5 degrees C. Cell attachment was followed by light microscopy by taking samples from the culture medium every 30 min. Urea and protein syntheses by microcarrier-attached hepatocytes were determined by standard techniques. Nonswellable (highly cross-linked) hydrophilic PHEMA microcarriers did not support cell attachment and viability. However, swellable (low cross-linked) PHEMA microcarriers (pretreated in FBS) allowed high attachment and cell spreading. PHEMA microcarriers treated in dimethylaminoethylmethacrylate (DMAEMA) glow-discharge plasma also improved the cell attachment characteristics of the PHEMA microcarriers. The highest attachment efficiencies (immobilization yields) were observed with the biologically modified PHEMA microcarriers, especially modified with fibronectin. Metabolic activity, as estimated by urea and protein syntheses, was also higher in these microcarriers.

Stimuli-responsive properties of conjugates of N-isopropylacrylamide-co-acrylic acid oligomers with alanine, glycine and serine mono-, di- and tri-peptides.

A random oligomer of N-isopropylacrylamide (NIPAAm) and acrylic acid (AAc) with a AAc content of 3.1+/-0.19 mmol carboxylic acid groups per gram of the oligomer and with a number average molecular weight of 1400 was synthesised by a free radical polymerisation using AIBN in DMF. Then, mono-, di-, and tri-peptide conjugates of this oligomer were prepared by using carboxyl-ends-protected (with methyl ester hydrochloride) forms of alanine, glycine and serine, with a water-soluble carbodiimide. 95, 93, and 31% of the carboxylic acids were conjugated (loaded) at the first step (mono-peptides) with glycine, alanine and serine, respectively. At the second step, percentage of the conjugation of carboxylic acid groups with glycine, alanine and serine were between 99 and 80, 68 and 100, and 21 and 58%, respectively, while the third amino acids were attached to only 21-64% of the carboxylic acids available on the conjugate chains. A decrease was observed in the lower critical solution temperatures (LCSTs) of the amino acid conjugates at pH 4.0 compared with the unconjugated oligomer, which has LCST at 37.7 degrees C at the same pH. LCSTs of di- and tri-peptide conjugates at pH 4.0 were in the range of 38.4-43.3 degrees C, and 42.6-50.8 degrees C, respectively. At pH 7.4, LCSTs of the mono- and di-peptide conjugates were observed in the range of 41.6-43.9 degrees C, and 46.2-60.2 degrees C, respectively, while the co-oligomer at pH 7.4 did not show a LCST up to 60 degrees C. Tri-peptide conjugates did not display LCST at pH 7.4, except the one with glycine-alanine-serine sequence.

Dye-ligand affinity systems.

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

Adsorption of heavy metal ions onto dithizone-anchored poly (EGDMA-HEMA) microbeads.

The dithizone-anchored poly (EGDMA-HEMA) microbeads were prepared for the removal of heavy metal ions (i.e. cadmium, mercury, chromium and lead) from aqueous media containing different amounts of these ions (25-500 ppm) and at different pH values (2.0-8.0). The maximum adsorptions of heavy metal ions onto the dithizone-anchored microbeads from their solutions was 18.3, Cd(II); 43.1, Hg(II); 62.2, Cr(III) and 155.2 mg g(-1) for Pb(II). Competition between heavy metal ions (in the case of adsorption from mixture) yielded adsorption capacities of 9.7, Cd(II); 28.7, Hg(II); 17.6, Cr(III) and 38.3 mg g(-1) for Pb(II). The same affinity order was observed under non-competitive and competitive adsorption, i.e. Cr(III)>Pb(II)>Hg(II)>Cd(II). The adsorption of heavy metal ions increased with increasing pH and reached a plateaue value at around pH 5.0. Heavy metal ion adsorption from artificial wastewater was also studied. The adsorption capacities are 4.3, Cd(II); 13.2, Hg(II); 7.2, Cr(III) and 16.4 mg g(-1) for Pb(II). Desorption of heavy metal ions was achieved using 0.1 M HNO(3). The dithizone-anchored microbeads are suitable for repeated use (for more than five cycles) without noticeable loss of capacity.

Novel dye-attached macroporous films for cadmium, zinc and lead sorption: Alkali Blue 6B-attached macroporous poly(2-hydroxyethyl methacrylate).

Alkali Blue 6B-attached poly(2-hydroxyethyl methacrylate) (poly(HEMA)) microporous films were investigated as chelate forming sorbents for heavy metal removal. Poly(HEMA) microporous films were prepared by UV-initiated photo-polymerization of HEMA in the presence of an initiator (azobisisobutyronitrile (AIBN)). Alkali Blue 6B was attached covalently. These films with a swelling ratio of 58%, and carrying 14.8 mmol Alkali Blue 6B m(-2) which were then used in the removal of Cd(II), Zn(II) and Pb(II) from aqueous media. Adsorption rates were very high, equilibrium was achieved in about 30 min. The maximum adsorption of heavy metal ions onto the Alkali Blue 6B-attached films were 41.4 mmol m(-2) for Cd(II), 52.4 mmol m(-2) for Zn(II), and 64.5 mmol m(-2) for Pb(II). When the heavy metal ions competed during the adsorption from a mixture the adsorption values for Cd(II), Zn(II) and Pb(II) were quite close. Heavy metal ions were desorbed by using 0.1 M HNO(3). A significant amount of the adsorbed heavy metal ions (up to 95%) could be desorbed in 30 min. Repeated adsorption/desorption cycles showed the feasibility of these novel dye-attached microporous films for heavy metal removal.

Functional assessment of human gastrointestinal tract using 99Tcm-latex particles.

99Tcm-latex particles (LP) is a newly developed radiopharmaceutical for the evaluation of the gastrointestinal (GI) tract. Following oral ingestion, it passes through the entire GI tract undissolved. The purpose of the present investigation was to introduce the clinical application of 99Tcm-LP. A group of 11 healthy volunteers was studied. Following a 12 h fast, the subjects were given 150 ml of water containing 37 MBq of 99Tcm-LP orally. Dynamic images up to 2 h were recorded to document temporal progression of radioactivity. Static images of the abdomen and whole body were taken at intervals. In normal subjects the tracer arrival times at the caecum and sigmoid colon were 3.2 +/- 0.9 and 11.2 +/- 3.2 h, respectively. The average t1/2 value for gastric emptying, 50% colonic filling and small bowel transit time were found to be 21.6 +/- 5.6, 233 +/- 72 and 211 +/- 66.4 min, respectively. In conclusion, 99Tcm-LP has the potential of providing functional information of the GI tract.

Coating of silicone-based impression materials in a glow-discharge system by acrylic acid plasma.

OBJECTIVES: The main purpose of this study was to demonstrate an increase in the wettability of silicone-based impression materials after coating them with a hydrophilic film in a glow-discharge system. METHODS: Two vinyl polysiloxane impression materials, Extrude (Kerr) and Accuflex (GC America Inc.) were used. Impression specimens were treated in a glow-discharge reactor at a radio frequency of 13.56 MHz at different discharge powers (5-20 W) and exposure times (5-60 min). Surface analysis of the specimens was done by FTIR. Surface contact angles were obtained by a captive-bubble method. These results were analyzed by ANOVA and Duncan's Multiple Range test (p < 0.05). The total number of voids on the die stone casts was observed microscopically. Linear dimensional accuracy, detail reproducibility, and surface hardness of the die stone casts were also determined. A Student t-test was performed for statistical analysis of these parameters (p > 0.05). RESULTS: FTIR spectra indicated that the number of hydroxyl groups on the surfaces increased (p > 0.05) because of the glow-discharge treatment. Contact angle measurements showed an increase (p < 0.05) in surface hydrophilicity. Total void formation in the stone casts decreased. There were no significant differences in the linear dimensional accuracy, detail reproducibility, and hardness, before and after glow-discharge treatment (p > 0.05). SIGNIFICANCE: It was concluded that the surface wettability of the impression materials may be increased by plasma deposition, and therefore, the formation of voids was reduced in the stone casts.

Optimization of urease immobilization onto non-porous HEMA incorporated poly(EGDMA) microbeads and estimation of kinetic parameters.

Jack bean urease (urea aminohydrolase, EC 3.5.1.5) was immobilized onto modified non-porous poly(ethylene glycol dimethacrylate/2-hydroxy ethylene methacrylate), (poly(EGDMA/HEMA)), microbeads prepared by suspension copolymerization for the potential use in hemoperfusion columns, not previously reported. The conditions of immobilization; enzyme concentration, medium pH, substrate and ethylene diamine tetra acetic acid (EDTA) presence in the immobilization medium in different concentrations, enzyme loading ratio, processing time and immobilization temperature were investigated for highest apparent activity. Immobilized enzyme retained 73% of its original activity for 75 days of repeated use with a deactivation constant kd = 3.72 x 10(-3) day(-1). A canned non-linear regression program was used to estimate the intrinsic kinetic parameters of immobilized enzyme with a low value of observable Thiele modulus (phi < 0.3) and these parameters were compared with those of free urease. The best-fit kinetic parameters of a Michaelis-Menten model were estimated as Vm = 3.318 x 10(-4) micromol/s mg bound enzyme protein, Km = 15.94 mM for immobilized, and Vm = 1.074 micromol NH3/s mg enzyme protein, Km = 14.49 mM for free urease. The drastic decrease in Vm value was attributed to steric effects, conformational changes in enzyme structure or denaturation of the enzyme during immobilization. Nevertheless, the change in Km value was insignificant for the unchanged affinity of the substrate with immobilization. For higher immobilized urease activity, smaller particle size and concentrated urease with higher specific activity could be used in the immobilization process.

Investigation of ascorbate-Cu (II) induced cleavage of DNA by scanning tunneling microscopy.

Scanning Tunneling Microscopy (STM) was used for the investigation of oxidative DNA damage. A PCR amplified fragment of human beta-globin gene was used as a model for time dependent cleavage reaction by ascorbate and copper. Cleavage reactions were carried out in a medium containing 0.5 microgram/20 microliters DNA, 20 nM Tris-HC1 pH, 7.8 and ascorbate-Cu (II) in the final concentrations of 1 mM and 30 microM, respectively. The mixtures were incubated at 37 degrees C for 5, 15 and 30 min. For STM studies, 3 pg/5 microliters DNA samples were deposited on the gold coated mica and dried in a water flow vacuum drier. The STM was operated in air at atmospheric pressure with a tip-to-substrate bias of 100 mV and tunneling currents of < 10 pA. Etched tips of Pt/Ir wires were used in a constant current mode. The degradated DNA structure can be distinguished from the intact DNA and the sizes of the degradation products can be identified in the STM micrographs. The size of fragments decreased from approximately 3000 A to 34 A in ascorbate-Cu (II) medium, after 30 min of incubation.

Interactions of DNA with fluorescent dyes: by scanning tunneling microscopy.

Genomic DNA was obtained from peripheral blood samples of healthy volunteers and interacted with two fluorescent dyes (i.e. Hoechst 33,258 and ethidium bromide) in aqueous media. These media containing DNA-dye complexes deposited on the gold coated mica surfaces. Then, STM images were obtained in which the STM was operated in air at atmospheric pressure with a tip-to-substrate bias voltage of 250-1000 mV (sample positive) and the tunneling currents in the range of 10-20 pA by using etched tips of Pt/Ir, in constant current mode. Both dyes from molecular clusters on DNA. While, the Hoechst molecules were observed on the DNA chains at regular distances, the ethidium bromide molecular clusters did not.

Blood plasma proteins on polyurethane and alkylsiloxane plasma-treated polyurethane surfaces. Dynamic approach by stimulus-response technique. Part 2. Evaluation of adsorption data by moment technique.

In this study, interactions of blood proteins (i.e. albumin and fibrinogen) with polyurethane biomaterial surfaces were investigated in an in vitro bead column test circuit using a stimulus-response technique. The dynamic sorption process of radiolabelled proteins on the surfaces was followed by detecting the radioactivity at the exit stream of the column, which was the response of a pulse stimulus at the inlet. The mathematical model was described and solved using 'parameter estimation by cybernetic moment technique', and the adsorption rate constants of plasma proteins on different biomaterial surfaces were calculated. By evaluation of the response curves with standard and cybernetic moment techniques, the following results were obtained. Albumin and fibrinogen adsorption is competitive, and the competition is strongly dependent upon the surface characteristics of the biomaterial. Preadsorption or preferential adsorption of albumin decreases the fibrinogen adsorption, and therefore increases the biocompatibility of material surface. Adsorption of blood plasma proteins are irreversible. The moment technique can also be used for the evaluation of stimulus-response data of biological systems, to determine the process parameters.