Precipitation of calcium phosphate in the presence of albumin on titanium implants with four different possibly bioactive surface preparations. An in vitro study

The aim of the present study was to compare the nucleating behaviour on four types of bioactive surfaces by using the simulated body fluid (SBF) model with the presence albumin. Titanium discs were blasted (B) and then prepared by alkali and heat treatment (AH), anodic oxidation (AO), fluoridation (F), or hydroxyapatite coating (HA). The discs were immersed in SBF with 4.5 mg/ml albumin for 3 days, 1, 2, 3 and 4 weeks and analysed with scanning electron microscopy/energy dispersive X-ray analysis (SEM/EDX) and X-ray photoelectron spectroscopy (XPS). Topographic surface characterisation was performed with a contact stylus profilometer. The results demonstrated that the bioactive surfaces initiated an enhanced calcium phosphate (CaP) formation and a more rapid increase of protein content was present on the bioactive surfaces compared to the blasted control surface. The observation was present on all bioactive surfaces. The fact that there was a difference between the bioactive surfaces and the blasted control surface with respect to precipitation of CaP and protein content on the surfaces support the fact that there may be biochemical advantages in vivo by using a bioactive surface.     

In vivo evaluation of titanium implants coated with bioactive glass by pulsed laser deposition

During the past years, different techniques, like chemical treatment, plasma spraying, sputtering, enamelling or sol–gel; and materials, like metals, hydroxylapatite, calcium phosphates, among others, have been applied in different combinations to improve the performance of prostheses. Among the techniques, Pulsed Laser Deposition (PLD) is very promising to produce coatings of bioactive glass on any metal alloy used as implant. In this work the biocompatibility of PLD coatings deposited on titanium substrates was examined by implantation in vivo. Different coating compositions were checked to find the most bioactive that was then applied on titanium and implanted into paravertebral muscle of rabbit.     

Evaluation of polyethersulfone highflux hemodialysis membrane in vitro and in vivo

A new hemodialysis membrane manufactured by a blend of polyethersulfone (PES) and polyvinylpyrrolidone (PVP) was evaluated in vitro and in vivo. Goat was selected as the experimental animal. The clearance and the reduction ratio after the hemodialysis of small molecules (urea, creatinine, phosphate) for the PES membrane were higher in vitro than that in vivo. The reduction ratio of β₂-microglobulin was about 50% after the treatment for 4 h. The biocompatibility profiles of the membranes indicated slight neutropenia and platelet adhesion at the initial stage of the hemodialysis. Electrolyte, blood gas, and blood biochemistry were also analyzed before and after the treatment. The results indicated that PES hollow fiber membrane had a potential widely use for hemodialysis.     

Biocompatibility of polyhydroxybutyrate microspheres: in vitro and in vivo evaluation

Microspheres have been prepared from the resorbable linear polyester of β-hydroxybutyric acid (polyhydroxybutyrate, PHB) by the solvent evaporation technique and investigated in vitro and in vivo. Biocompatibility of the microspheres has been proved in tests in the culture of mouse fibroblast cell line NIH 3T3 and in experiments on intramuscular implantation of the microspheres to Wistar rats for 3 months. Tissue response to the implantation of polymeric microspheres has been found to consist in a mild inflammatory reaction, pronounced macrophage infiltration that increases over time, involving mono- and poly-nuclear foreign body giant cells that resorb the polymeric matrix. No fibrous capsules were formed around polymeric microparticles; neither necrosis nor any other adverse morphological changes and tissue transformation in response to the implantation of the PHB microparticles were recorded. The results of the study suggest that polyhydroxybutyrate is a good candidate for fabricating prolonged-action drugs in the form of microparticles intended for intramuscular injection.     

Immobilized-OPG-Fc on a titanium surface inhibits RANKL-dependent osteoclast differentiation in vitro

The purpose of the present study was to examine the effect of osteoprotegerin (OPG)-Fc fusion protein immobilized on a titanium surface on the initial differentiation of osteoclast precursor RAW264.7 cells. These cells were cultured on titanium specimens over which OPG-Fc was immobilized. The enhancement of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expression in RAW264.7 cells exposed to receptor activator of NF-κB ligand (RANKL) stimulation on OPG-Fc-coated titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on titanium specimens without immobilized OPG-Fc (ANOVA, P < 0.01). Preincubation of OPG-Fc-coated titanium, in a medium supplemented with 10% fetal bovine serum at 37°C for two days before the cells were seeded, had no significant effect on the decrease in mRNA expression (ANOVA, P < 0.01). Taken together, these results indicate that OPG-Fc immobilized on a titanium surface blocks the differentiation of RAW264.7 cells induced by RANKL stimulation.     

The controlling biodegradation of chitosan fibers by N-acetylation in vitro and in vivo

Aim In the present study, we investigated the biodegradation of the fibers of chitosan and its acetylated derivatives in vitro and in vivo. Methods A series of chitosan fibers, with acetylation degrees of 7.7%, 21.6%, 40.9%, 61.2%, 82.5% and 93.4%, were obtained by acetylating chitosan filament with acetic anhydride, and were investigated by FT-IR analysis, elemental analysis and scanning electron microscopy analysis. Results The in vitro experimental data indicated that the degradation rate of chitosan fiber was strongly dependent on the degree of acetylation, and the degradation rate increased with an enhancement of the acetylation degree of chitosan fibers. In vivo degradation experiment evaluated by light microscopy as well as scanning electron microscopy, was studied by implanting the fibers between the two nerve stumps of the rat sciatic nerve gap (6 months). The findings demonstrated that acetylation degree could influence the degradation rate of chitosan fibers in vivo. Conclusion These results suggested that acetylated chitosan (chitin) fibers were more biodegradable than chitosan and the biodegradation rate of chitin fiber can be controlled to desirable extent by the variation of acetylation degree.     

In vitro and in vivo degradation of poly(l-lactide-co-glycolide) films and scaffolds

Poly(l-lactide-co-glycolide) (PLGA) was synthesized using a biocompatible initiator, zirconium acetylacetonate. In vitro and in vivo degradation properties of PLGA films (produced by solvent casting, 180 μm thick) and PLGA scaffolds (produced by an innovated solvent casting and particulate leaching, 3 mm thick) were evaluated. The samples were either submitted for degradation in phosphate buffered saline (PBS) at 37 °C for 30 weeks, or implanted into rat skeletal muscles for 1, 4, 12, 22 and 30 weeks. The degradation was monitored by scanning electron microscopy, atomic force microscopy, weight loss, and molecular weight changes (in vitro), and by microscopic observations of the materials’ morphology after histological staining with May-Grunwald-Giemsa (in vivo). The results show that the films in both conditions degraded much faster than the scaffolds. The scaffolds were dimensionally stable for 23 weeks, while the films lost their integrity after 7 weeks in vitro. The films’ degradation was heterogenous—degradation in their central parts was faster than in the surface and subsurface regions due to the increased concentration of the acidic degradation products inside. In the scaffolds, having much thinner pore walls, heterogenous degradation due to the autocatalytic effect was not observed.     

Bone response to surface modified titanium implants – studies on the tissue response after 1 year to machined and electropolished implants with different oxide thicknesses

The bone formation around titanium implants with varied surface properties was investigated after 1 year in rabbits. Machined and electropolished samples with and without thick, anodically formed surface oxides were prepared, surface characterized and inserted in the cortical bone of rabbits. Scanning electron microscopy, scanning Auger electron spectroscopy and atomic force microscopy revealed marked differences in oxide thickness, surface topography and roughness, but no significant differences in surface chemical composition between the different groups of implants. Light microscopic morphology and morphometry showed that all implants were in contact with bone and had a large proportion of bone within the threads. There were no significant differences between the differently prepared implant groups. Our study shows that a high degree of bone contact and bone formation is achieved after 1 year with titanium implants which are modified with respect to oxide thickness and surface topography. There is no indication that a reduction of surface roughness, which in the initial phase decreases the rate of bone formation, had any influence on the amount of bone after 1 year in rabbit cortical bone.     

Human tissue allograft processing: impact on in vitro and in vivo biocompatibility

This work investigates the impact of chemical and physical treatments on biocompatibility for human bone/tendon tissues. Nontreated and treated tissues were compared. In vitro testing assessed indirect and direct cytotoxicity. Tissues were subcutaneously implanted in rats to assess the immunological, recolonization, and revascularization processes at 2–4 weeks postimplantation. No significant cytotoxicity was found for freeze-dried treated bones and tendons in comparison to control. The cellular adhesion was significantly reduced for cells seeded on these treated tissues after 24 h of direct contact. A significant cytotoxicity was found for frozen treated bones in comparison to freeze-dried treated bones. Tissue remodeling with graft stability, no harmful inflammation, and neo-vascularization was observed for freeze-dried chemically treated bones and tendons. Frozen-treated bones were characterized by a lack of matrix recolonization at 4 weeks postimplantation. In conclusion, chemical processing with freeze-drying of human tissues maintains in vitro biocompatibility and in vivo tissue remodeling for clinical application.     

In vitro and in vivo biological performance of collagen-chitosan/silicone membrane bilayer dermal equivalent

Skin loss or damage affects severely the life quality of human being and can even cause death in many cases. We report here a bilayer dermal equivalent (BDE) composed of collagen-chitosan porous scaffold and silicone membrane, which can effectively induce the regeneration of dermis in an animal model of full thickness skin loss. The in vitro biosecurity test showed that the BDE had no cytotoxicity, and no remarkable sensitization and irritability. In vitro cell culture proved that the BDE had good biocompatibility to support the proliferation of fibroblasts. Animal test was performed on Bama miniature pig skin. Gross view and histological sections found plenty of fibroblasts and extracellular matrix in the regenerative scaffold after transplantation of the BDE for 4 weeks. Immunohistochemistry results proved that the BDE has the ability to support the angiogenesis of the regenerated dermis. All these results indicate that the BDE might be a promising equivalent in treating dermal loss.     

Bioactivity of ceramic–polymer composites with varied composition and surface topography

HAPEX trade mark (40 vol % hydroxyapatite in a high-density polyethylene matrix) and AWPEX (40 vol % glass-ceramic apatite-wollastonite in a high-density polyethylene matrix) are composites designed to provide bioactivity and to match the mechanical properties of human cortical bone. HAPEX trade mark has had clinical success in middle ear and orbital implants, and there is great potential for further orthopaedic applications of these materials. However, more detailed in vitro investigations must be performed to better understand the biological interactions of the composites. In this study, the bioactivity of each material was assessed. Specifically, the effects of controlled surface topography and ceramic filler composition on apatite layer formation in acellular simulated body fluid (SBF) with ion concentration similar to those of human blood plasma were examined. Samples were prepared as 1 x 10 x 10 mm(3) tiles with polished, roughened or parallel-grooved surface finishes, and were incubated in 20 ml of SBF at 36.5 degrees C for one, three, seven or 14 days. The formation of an apatite layer on the composite surface after immersion was demonstrated by thin-film X-ray diffraction, environmental scanning electron microscopy and energy dispersive X-ray analysis. Variations in sample weight and solution pH over the period of incubation were also recorded. Significant differences were found between the two materials tested, with greater bioactivity in AWPEX than HAPEX trade mark. Results also showed surface topography to be important, with rougher samples correlated to earlier apatite formation. Osteoblast-like cells proliferated favourably on both composite materials, with many filopodia connections, preferential attachment to ceramic particles and contact guidance effects evident.     

Degradative and mechanical properties of a novel resorbable plating system during a 3-year follow-up in vivo and in vitro

We tested the tissue reactions and mechanical strength of a novel biodegradable craniomaxillofacial plating system, Inion CPS™, in the course of degradation. Plates and screws composed of l-lactide, d-lactide and trimethylene carbonate were implanted to the mandible and dorsal subcutis of 12 sheep. The animals were sacrificed at 6–156 weeks. Histological evaluation was done using paraffin and methylmetacrylate techniques. Degradative and mechanical properties during the follow-up were measured both of in vivo and in vitro implants. In light microscopy, the in vivo implant material began to fragment at 52 weeks and could not be detected at 104 weeks. No significant foreign body reactions were seen in the mandibles. The dorsal subcutis disclosed mild reactions, which were, however, not of clinical significance. The implants in vitro maintained their entire mass for 26 weeks and lost 63–80% of the mass by week 104. The inherent viscosity of the implants in vitro and in vivo diminished uniformly. The screws retained their shear strength for 12–16 weeks. The plates maintained their tensile strength for at least 6 weeks. The maximum capacity of the plates in 3-point bending tests diminished gradually by 87% in 26 weeks. In conclusion, the plates and screws examined maintain adequate strength for the healing period of a bone fracture or osteotomy, producing no harmful foreign body reactions. Dr Nieminen is a consultant for Inion Ltd., while the other co-authors do not have any conflicts of interest. Inion Ltd. has financed the costs related to the study sheep, including their housing.     

In vitro and in vivo sustained release of exenatide from vesicular phospholipid gels for type II diabetes

Diabetes is a chronic disease that requires daily treatment to maintain a stable blood glucose level. Sustained-release formulations can thus benefit the treatment of diabetes by reducing the repeated administration of therapeutics. Our study aimed to develop a sustained-release platform for exenatide that is biocompatible and capable of mass production. Vesicular phospholipid gels (VPGs) are semisolid phospholipid dispersions with controlled release profiles. Exenatide-VPGs prepared via simple magnetic stirring showed excellent biocompatibility with an average particle size of about 15?μm after redispersion. VPGs were shown to achieve sustained release for up to 21 days in vitro with no obvious burst effect. The in vivo release study showed that VPGs sustained the release of the exenatide for up to 11 days. Moreover, after subcutaneous injection of the exenatide-VPGs in the diabetic rats, the hypoglycemic effect lasted for 10 days compared with exenatide solution. In sum, the exenatide-VPGs system represents a promising sustained-release formulation for exenatide with a long-acting therapeutic efficacy in vivo.     

In vitro macrophage uptake and in vivo biodistribution of PLA–PEG nanoparticles loaded with hemoglobin as blood substitutes: effect of PEG content

The aim of the present work is to investigate the effect of PEG content in copolymer on physicochemical properties, in vitro macrophage uptake, in vivo pharmacokinetics and biodistribution of poly(lactic acid) (PLA)–poly(ethylene glycol) (PEG) hemoglobin (Hb)-loaded nanoparticles (HbP) used as blood substitutes. The HbP were prepared from PLA and PLA–PEG copolymer of varying PEG contents (5, 10, and 20 wt%) by a modified w/o/w method and characterized with regard to their morphology, size, surface charge, drug loading, surface hydrophilicity, and PEG coating efficiency. The in vitro macrophage uptake, in vivo pharmacokinetics, and biodistribution following intravenous administration in mice of HbP labeled with 6-coumarin, were evaluated. The HbP prepared were all in the range of 100–200 nm with highest encapsulation efficiency 87.89%, surface charge −10 to −33 mV, static contact angle from 54.25° to 68.27°, and PEG coating efficiency higher than 80%. Compared with PLA HbP, PEGylation could notably avoid the macrophage uptake of HbP, in particular when the PEG content was 10 wt%, a minimum uptake (6.76%) was achieved after 1 h cultivation. In vivo, besides plasma, the major cumulative organ was the liver. All PLA–PEG HbP exhibited dramatically prolonged blood circulation and reduced liver accumulation, compared with the corresponding PLA HbP. The PEG content in copolymer affected significantly the survival time in blood. Optimum PEG coating (10 wt%) appeared to exist leading to the most prolonged blood circulation of PLA–PEG HbP, with a half-life of 34.3 h, much longer than that obtained by others (24.2 h). These results demonstrated that PEG 10 wt% modified PLA HbP with suitable size, surface charge, and surface hydrophilicity, has a promising potential as long-circulating oxygen carriers with desirable biocompatibility and biofunctionality.     

In vitro and in vivo characterization of fast dissolving tablets containing gliquidone–pluronic solid dispersion

Abstract

The main objective of this study is to increase the dissolution rate of gliquidone using its solid dispersions with pluronic F-68 by solvent evaporation method. The solid dispersion of the drug with pluronic at ratio 1:3 showed the highest dissolution efficiency (50.7%) after 10?min, so it was incorporated in fast dissolving tablets. Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC) were used to study the interaction between gliquidone and pluronic in the solid state. The FTIR spectroscopic studies revealed no chemical interaction between the drug and pluronic, while the DSC results indicated the amorphous state of gliquidone in the solid dispersion. A 3² full factorial design was used to study the effect of varying concentrations of croscarmellose and sodium starch glycolate as superdisintegrants on the disintegration time and the percentage released after 10?min. The optimized formula showed a disintegration time of 39.1?±?1.2?s and 85.43%?±?5.16% released after 10?min and was selected for the in-vivo studies in rabbits. The selected formula showed significant enhancement of gliquidone bioavailability, about 1.8 times compared with the commercial Glurenor tablets.     

In vivo behaviour of two different biphasic ceramic implanted in mandibular bone of dogs

Alloplastic calcium phosphate bone substitutes such as hydroxyapatite (HA) and tricalcium phosphate (TCP) have been studied extensively due to their composition closely resembling the inorganic phase of bone tissue. On the same way, by manipulating the HA/TCP ratio it may be possible to change the substitution rate and the bioactivity of these materials, an advantage which has brought them to clinical use in oral and orthopaedic surgery. In this work, we evaluated the histological response in bone of two biphasic calcium phosphate ceramics by varying the proportion of their components. All premolars of 6 beagle dogs were removed from both sides of the mandible. Three months later, four cylinders composed of 85% HA and 15% β-TCP (BCP 1) were implanted in the right side of mandible and other four cylinders composed of 15% HA and 85% β-TCP (BCP 2) were implanted in the left side of mandible of dogs for 4, 12 ad 26 weeks, respectively. Two dogs were used in each time point. The histological study indicated that both biphasic ceramic were biocompatible. The earlier and more quantity of bone formed in BCP 2 than in BCP 1 suggested that the first one had a higher osteoinductive potential than the second one in mandibular bone. The resorption of the phosphate phase and the subsequent migration of bone into the resorbed portions were detected in both biphasic ceramics although two processes appeared faster in BCP 2 than in BCP 1. These dates conclude that varying the components of our biphasic ceramic we improve its osteoinductive potential.     

In vivo and in vitro response to electrochemically anodized Ti-6Al-4V alloy

Tissues’ reactions to metals depend on a variety of properties of the metal, most notably surface structure. Anodizing has been shown to alter the surface properties of metal, thus eliciting a change in the biocompatibility of the metal. In order to evaluate the biocompatibility of unoxidized titanium alloy (Ti-6Al-4V) and anodized titanium alloy samples, the samples were implanted in murine abdominal subcutaneous tissues, and maintained for 2 and 4 weeks. The reaction of the abdominal subcutaneous connective tissues to the samples was then assessed. Fibrous connective tissue capsules were observed around the vicinity of the sample, and these capsules were shown to harbor fibroblasts, fibrocytes, and other cells, including neutrophils, macrophages, and giant multinucleated cells. The average thickness of the fibrous capsules observed around the anodized alloy samples was less than that of the capsules seen around samples of the unoxidized titanium alloy. Blood was obtained from the tails of the experimental mice, and blood cell analyses were conducted in order to assess the levels of leukocytes, red blood cells, and thrombocytes. The blood analysis results of the unoxidized control group and treatment group were all within normal ranges. In addition, the biocompatibility of the titanium alloy samples was evaluated using cell culture techniques. The numbers of MG-63 cells cultured on oxidized samples tended to be greater than those in the controls; however, these increases were not statistically significant. The alkaline phosphatase activity of the sample oxidized at 310 V evidenced significantly higher activity than was observed in the control group. These results indicate that the anodized Ti-6Al-4V alloy will be of considerable utility in biomedical applications.     

In vitro and in vivo studies on the complexes of glipizide with water-soluble β-cyclodextrin-epichlorohydrin polymers

The purpose of this study was to evaluate the potential of a newly modified cyclodextrin derivative, water-soluble β-cyclodextrin-epichlorohydrin polymer (β-CDP), as an effective drug carrier to enhance the dissolution rate and oral bioavailability of glipizide as a poorly water-soluble model drug. Inclusion complexes of glipizide with β-CDP were prepared by the co-evaporation method and characterized by phase solubility, dissolution, and differential scanning calorimetry. The solubility curve was classified as type A(L), which indicated the formation of 1:1 complex between glipizide and β-CDP. β-CDP had better properties of increasing the aqueous solubility of glipizide compared with HP-β-CD. The dissolution rate of drug from the β-CDP complexes was significantly greater than that of the corresponding physical mixtures indicating that the formation of amorphous complex increased the solubility of glipizide. Moreover, the increment in drug dissolution rate from the glipizide/β-CDP systems was higher than that from the corresponding ones with HP-β-CD, which indicated that β-CDP could provide greater capability of solubilization for poorly soluble drugs. Furthermore, in vivo study revealed that the bioavailability of glipizide was significantly improved by glipizide /β-CDP inclusion complex after oral administration to beagle dogs.     

Increase or decrease in stability after nucleotomy? Conflicting in vitro and in vivo results in the sheep model

Nucleotomy is a common surgical procedure to treat disc herniations. The potential occurrence of segmental instability after surgery, however, is suspected to necessitate re-operation and fusion. Although in vitro studies support the theory of destabilization after nucleotomy, a prior, in-house animal study contrarily revealed an increase in stability after surgery. To identify which structural compartment of the motion segment is decisive for increased stability after nucleotomy in vivo, the flexibilities of ovine motion segments were measured after different stepwise reductions at the anterior and posterior spinal column. Different test groups were used in which nucleotomy had been performed during surgery in vivo and under isolated in vitro conditions, respectively. In accordance with expectations, in vitro nucleotomy on ovine motion segments significantly increased flexibility. By contrast, nucleotomy significantly decreased flexibility 12 weeks after surgery. After removal of the posterior structures, however, the differences in flexibility diminished. The present results thus suggest that it might not exclusively be the trauma to the intervertebral disc during surgery which is decisive for post-operative stability, but rather adaptive mechanisms in the posterior structures. Therefore, care should be taken to minimize the damage to the posterior structures in the course of the surgical approach, which more likely compromises stability.     

Microstructural and tribological characterization of air plasma sprayed nanostructured alumina–titania coatings deposited with nitrogen and argon as primary plasma gases

Air plasma sprayed nanostructured Al₂O₃–13wt%TiO₂ coatings were deposited as a function of critical plasma spray parameter (CPSP), defined as the ratio of arc power to primary gas flow rate, using nitrogen and argon as the primary plasma gases. Microstructural features including percentage of α-Al₂O₃ phase, percentage of partially melted/unmelted regions, microhardness, and wear characteristics were evaluated for the deposited coatings. Effect of CPSP on microstructural and wear characteristics of coatings deposited with nitrogen was found to be relatively small. In contrast, significant effect of CPSP on coating characteristics was found for coatings deposited with argon. In wear tests, while strong effect of normal load on weight loss was observed for coatings deposited with nitrogen, weight loss for coatings deposited with argon was nearly independent of applied normal load, at least for coatings deposited at the highest CPSP.