A new osteoblast cell culture system for standardized testing of biomaterials

For in vitro testing of new biomaterials cultured fibroblasts are employed. In the case of the agar diffusion test survival of cells is involved in the presence of the material to be tested. Further statements on the biological effects of a biomaterial require the use of cell cultures adapted to the tissue concerned and the underlying problem being investigated. In the present study, an osteoblast cell culture system with which implant surfaces in contact with bone can be tested as required by the relevant standards is described. Test bodies made of titanium, polystyrene or copper were used in the conventional agar diffusion test, and were also overgrown with fibroblasts or a cell line of foetal human osteoblasts. For the agar diffusion test, the test criterion was the extent of the inhibition area on staining with neutral red, while for the overgrowth, the mean cell diameter and the number of cells was employed. The phenotype of the osteoblast cell line was determined immunohistochemically by means of alkaline phosphatase or immunohistologically by means of collagen I and osteocalcin. Calcification was demonstrated using the v. Kossa stain. In the case of the osteoblasts, a differentiation of a collagen I and alkaline phosphatase-positive phenotype over an osteocalcin-positive phenotype to an increase in calcium deposition was shown. As in the case of the agar diffusion test, direct overgrowth also revealed no cytotoxic effect for titanium and polystyrene. In contrast, a cytotoxic effect consisting in a decrease in the number of cells and also a left shift in the size distribution was observed for copper. The standard deviations of the individual tests were less for overgrowth than for the agar diffusion test. The culture system for osteoblast cells thus meets the criteria of the EN/DIN 30993-5 in terms of the quality and accuracy of the results obtained. In addition to excluding direct cytotoxicity, this test system offers a new possibility of examining the influence of the material on cell growth. Consequently, it permits a repeatable examination of proliferation and differentiation of the osteoblasts on each material surface.     

A numerical study of the effect of drive level on the intensity loss from an ultrasonic beam

Knowledge of the spatial distribution of intensity loss from an ultrasonic beam is vital to bioeffect predictions such as heating and streaming. A method for calculating the distribution of intensity loss over a finite-amplitude ultrasonic beam is described. The technique demonstrates that the location of peak intensity loss varies considerably with drive level for a plane circular source, as does the overall distribution of intensity loss across the beam. The effects are shown to be less pronounced but still significant for a focused source.     

A study on measurement of gas-liquid interfacial area in a dispersed gas injection system

The gas-liquid interfacial area in a dispersed gas injection system was estimated by a chemical method and measured directly by an electroresistivity probe method. In the chemical method, the gas-liquid interfacial area was obtained by plotting CO₂ absorption rate in 0.1 mol/L KOH solution based on the gas absorption theory. In the electroresistivity probe method, local gas holdup, bubble rising velocity, and local bubble diameter were measured in the same solution into which air was injected upwardly. Overall mean values of gas holdup and bubble diameter in the vessel were obtained by means of statistical treatments. The specific interfacial area was calculated from these data. Measurements were done in a cylindrical water vessel under different conditions of gas flow rate and nozzle diameter. The measured values of the specific interfacial area by the electroresistivity probe method were compared with those by the chemical method and showed good agreement. H.K. PARK, formerly Graduate Student, Seoul National University.     

A peptide that inhibits hydroxyapatite growth is in an extended conformation on the crystal surface

Proteins play an important role in the biological mechanisms controlling hard tissue development, but the details of molecular recognition at inorganic crystal interfaces remain poorly characterized. We have applied a recently developed homonuclear dipolar recoupling solid-state NMR technique, dipolar recoupling with a windowless sequence (DRAWS), to directly probe the conformation of an acidic peptide adsorbed to hydroxyapatite (HAP) crystals. The phosphorylated hexapeptide, DpSpSEEK (N6, where pS denotes phosphorylated serine), was derived from the N terminus of the salivary protein statherin. Constant-composition kinetic characterization demonstrated that, like the native statherin, this peptide inhibits the growth of HAP seed crystals when preadsorbed to the crystal surface. The DRAWS technique was used to measure the internuclear distance between two 13C labels at the carbonyl positions of the adjacent phosphoserine residues. Dipolar dephasing measured at short mixing times yielded a mean separation distance of 3.2 +/- 0.1 A. Data obtained by using longer mixing times suggest a broad distribution of conformations about this average distance. Using a more complex model with discrete alpha-helical and extended conformations did not yield a better fit to the data and was not consistent with chemical shift analysis. These results suggest that the peptide is predominantly in an extended conformation rather than an alpha-helical state on the HAP surface. Solid-state NMR approaches can thus be used to determine directly the conformation of biologically relevant peptides on HAP surfaces. A better understanding of peptide and protein conformation on biomineral surfaces may provide design principles useful for the modification of orthopedic and dental implants with coatings and biological growth factors that are designed to enhance biocompatibility with surrounding tissue.     

Chondrocyte-seeded hydroxyapatite for repair of large articular cartilage defects. A pilot study in the goat

The aim of this study was to evaluate the potential for restoration of a large cartilage defect in the goat knee with hydroxyapatite (HA) loaded with chondrocytes. Isolated chondrocytes were suspended in fibrin glue, seeded on top of the HA, and then the composite graft was implanted in the defect. After transplantation, cell behaviour, newly synthesised matrix and the HA-glue interface were assessed histologically after 2, 4, 12, 26 and 52 weeks. Special attention was paid to the incorporation process of HA in the subchondral bone and interactions between this biomaterial and the fibrin-glue-chondrocyte suspension. Chondrocytes in the glue proved to survive the transplantation procedure and produced new metachromatically stained matrix two weeks after implantation. The glue-cell suspension had penetrated the superficial porous structure of the HA. Four weeks after surgery, islands of hyaline-like cartilage were observed at the HA-glue interface. A layer of fibrous tissue was formed surrounding the HA graft, resulting in a relatively instable fixation of the HA in the defect. This instability of the graft in the defect, possibly together with early weight bearing, resulted in a gradual loss of the newly formed hyaline cartilage-like repair tissue. Progressive resorption of the HA occurred without any sign of active bone remodelling from the host site. One year after surgery part of the defect which extended down to the cancellous bone had been predominantly restored with newly formed lamellar bone. Only small HA remnants were still present at the bottom of the original defect. Resurfacing of the joint had occurred with fibrocartilaginous repair tissue. The absence of adequate fixation capacity of the HA near the joint space resulted in a relative instability of the graft with progressive resorption. Therefore, HA is not a suitable biomaterial to facilitate the repair of large articular cartilage defects.     

A pulmonary vascularization study in pulmonary atresia with an interventricular defect in relation to the presence of a chromosome 22 deletion

BACKGROUND/AIMS: Bare sclera resection with and without use of mitomycin C and conjunctival autograft placement are three surgical techniques currently in use for the treatment of primary pterygium. The purpose of this study was to determine through a meta-analysis the risk for postoperative pterygium recurrence comparing the three surgical treatment modalities. METHODS: A search through Medline for randomised controlled clinical trials comparing at least two of the three surgical techniques in the treatment of primary pterygium, along with a hand search of all references in relevant papers, was conducted. All eligible clinical trials were graded for quality utilising the Detsky score; those studies with a score of 0.5 or greater were included. The main outcome measurements were the pooled odds ratios and 95% confidence intervals for the risk of pterygium recurrence. These were calculated utilising the Mantel-Haenszel method. RESULTS: Five eligible studies with an adequate quality score were retrieved, three comparing bare sclera resection with and without mitomycin C use, one comparing bare sclera resection with conjunctival autograft placement, and one comparing both. The pooled odds ratio for pterygium recurrence in patients who had only bare sclera resection was 6.1 (95% confidence intervals, 1.8 to 18.8) compared with the patients who had conjunctival autograft placement and 25.4 (9.0 to 66.7) compared with the patients who received mitomycin C. CONCLUSIONS: The odds for pterygium recurrence following surgical treatment of primary pterygium are close to six and 25 times higher if no conjunctival autograft placement is performed or if no intra/postoperative mitomycin C is used, respectively. Surgeons and clinical triallists should not be encouraged in the use of bare sclera resection as a surgical technique for primary pterygium.     

A quantitative study of the sintering and mechanical properties of hydroxyapatite/phosphate glass composites

Previous work has shown that small additions of a phosphate glass (CaO-P2O5) can significantly enhance the sinterability and strength of hydroxyapatite. However, there are no quantitative phase analyses available for these materials which would provide indicators of biocompatibility and resorbability. Similarly, there is little information available about the mechanical properties, especially with high glass additions. In this study, the effects of sintering hydroxyapatite with phosphate glass additions of 2.5, 5, 10, 25, and 50 wt.% are quantified. Each composition was sintered over a range of temperatures, and quantitative phase analysis was carried out using XRD. In addition, the microstructures were studied using RLOM and SEM, and mechanical properties (Vickers hardness, KIC, and MOR) measured. These results may be used to indicate which compositions and processing conditions may provide materials suitable for use in hard tissue replacement. Composites containing up to 10 wt.% glass additions formed dense HA/TCP composite materials possessing flexural strength and fracture toughness values up to 200% those of pure HA. The HA/TCP ratio was strongly dependent on the percentage glass addition. Higher glass additions resulted in composites containing beta-TCP together with large amounts of alpha- or beta-calcium pyrophosphate, and having similar mechanical strengths to pure HA.     

Endothelial cells in culture: an experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [3H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor-alpha and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selection and vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also in rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.     

A partial involvement of histamine in ultrasonically nebulized distilled water-induced bronchoconstriction in guinea-pigs

1. Inhalation of ultrasonically nebulized distilled water (UNDW) can induce bronchoconstriction only in asthmatics, but mechanisms of the response are not well known. We recently reported a guinea-pig model of UNDW-induced bronchoconstriction (UNDW-IB) in which UNDW induces bronchoconstriction when UNDW is inhaled 20 min after a challenge with aerosolized ovalbumin (OA) in passively sensitized, anaesthetized and artificially ventilated guinea-pigs. 2. To elucidate the role of histamine in the UNDW-IB, we examined the effects of antihistamines, diphenhydramine hydrochloride (DH) and chlorpheniramine maleate (CP), and measured histamine content in bronchoalveolar lavage fluid (BALF) in the animal model. 3. DH in doses of 0.1, 1.0 and 10 mg kg(-1) and CP in doses of 0.01, 0.1 and 1.0 mg kg(-1) administered intravenously 15 min after the OA challenge partially reduced the UNDW-IB at 1 and 2 min after the UNDW inhalation in a dose-dependent manner. Histamine content in BALF recovered 10 min after the UNDW inhalation following the OA provocation was significantly increased compared with that after saline inhalation and before the UNDW inhalation following the OA challenge. 4. Intravenous atropine in a dose of 0.5 mg kg(-1) or inhaled disodium cromoglycate in concentrations of 1 and 10 mg ml(-1) did not alter the UNDW-IB. 5. These results suggest that histamine is involved in part in the UNDW-IB in our animal model.     

A long-time presence of a foreign body in an episiotomy scar

OBJECTIVES: The object of our study was a long-time presence of a foreign body in an episiotomy scar. STUDY DESIGN: A case of 46-year old woman with a palpable tumour in a perineal region is described. RESULTS: This false tumour was caused by a surgical needle which was left in the body of the patient 19 years ago during the episiorrhaphy after the last labour. CONCLUSION: Because of a character and localisation of the change the method of choice was surgical treatment which completely eliminated all symptoms.     

Validation of precision-cut liver slices in dynamic organ culture as an in vitro model for studying CYP1A1 and CYP1A2 induction

The utilization of precision-cut liver slices in dynamic organ culture as an in vitro model was validated by comparing the induction of the biomarker responses following in vitro (rat liver slice) and in vivo exposure of rats to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The biomarker responses investigated were cytochrome P450s 1A1 and 1A2 (CYP1A1 and CYP1A2) mRNA, protein, and activities. Precision-cut rat liver slices were incubated in dynamic organ culture for 24 hr with medium containing 0.001-10 nM TCDD or medium without TCDD (control). The resultant mean TCDD concentration in the slices ranged from 19 to 80,925 ppt (wet wt), respectively. A concentration-dependent induction of CYP1A1 mRNA, protein, and activities and a more modest induction of CYP1A2 mRNA was observed in liver slices at all medium concentrations of TCDD. The O-demethylation of 7-methoxyresorufin, a marker for CYP1A2 activity, was induced at TCDD medium levels of 0.01 nM and greater, whereas a detectable increase in CYP1A2 protein occurred only at the higher concentrations. Comparable liver concentrations of TCDD (8-64,698 ppt wet wt) were achieved at 24 hr following a single in vivo exposure of rats to TCDD at doses ranging from 0.002 to 5 microg/kg po. Concentration-effect and dose-response relationships for induction of CYP1A1 and CYP1A2 were similar following in vitro and in vivo exposure to TCDD, although the magnitude of induction was greater for in vivo exposure. The data support the use of liver slices in dynamic organ culture for assessing the relative in vivo potency of a compound to induce CYP1A1 and CYP1A2. Human tissue can also be readily utilized in this in vitro model to predict the biological and toxicological effects of a given in vivo exposure to TCDD.     

A kinetic study of the coupled iron-ceruloplasmin catalyzed oxidation of ascorbate in the presence of albumin

Ascorbate is catalytically oxidized by a couple iron-ceruloplasmin system, the iron ions functioning as a red/ox cycling intermediate between ceruloplasmin and ascorbate. Serum albumin, an iron binding compound, was found to stimulate the ascorbate oxidation rate. It is proposed that ferrous ions react more rapidly with ceruloplasmin when they are bound to albumin. A Km value of 39 microM was estimated for Fe(2+)-albumin. Citrate and urate inhibit the iron-ceruloplasmin-dependent ascorbate oxidation by chelating ferric ions. In the presence of albumin only citrate reduced the oxidation rate, the observation suggesting the following order of iron binding ability: citrate > albumin > urate. Physiological aspects of the results have been discussed.     

A new culture system to study the metabolism of the intervertebral disc in vitro

STUDY DESIGN: This study determined whether entrapment of a rabbit intervertebral disc in alginate gel helped to promote the retention of normal metabolic activities by the nucleus pulposus and anulus fibrosus in tissue culture. OBJECTIVES: To establish an in vitro culture system to study the metabolism of the intervertebral disc as a whole integral organ. SUMMARY OF BACKGROUND DATA: In vitro studies of the metabolism of intervertebral discs have been scarce because of the difficulties involved in maintaining the integrity of the tissues, especially that of the nucleus pulposus, in culture medium. METHODS: Rabbit intervertebral discs were embedded in alginate gel and maintained in culture for as long as 1 month. At weekly intervals, experiments were performed to measure the rate of proteoglycan synthesis and to characterize proteoglycans newly synthesized by cells in the anulus fibrosus and nucleus pulposus. In addition, at these same time intervals, the contents of sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, and collagen in these two intervertebral disc tissues were measured to evaluate tissue integrity. Intervertebral discs cultured in medium alone were used as controls and analyzed in parallel. RESULTS: The anulus fibrosus and nucleus pulposus of intervertebral discs cultured in alginate gel sustained a higher rate of proteoglycan synthesis and maintained a higher content of extracellular matrix components than the respective controls at all times. CONCLUSIONS: This new alginate tissue culture system should prove useful for studying the metabolism of whole intervertebral discs.     

A comparative study of dynamic and static testing in abstinent alcoholics

The performance of 44 Finnish alcoholics was measured on a Vygotskian version of the Raven's Progressive Matrices and four traditional static tests (the Digit Symbol and Block Design subtests from the WAIS, Part B of the Trial Making Test, and the Embedded Figures Test). Of the static tests only the Block Design showed consistent congruence with the results of the learning potential test. The other static tests discriminated only partially from each other the groups which were formed according to learning potential. The most fruitful way to test alcoholics is probably to have a flexible combination of static and dynamic tests. We also need to have tests that are applicable to both static and dynamic testing.     

Moderate intensity exercise as an adjunct to standard smoking cessation treatment for women: A pilot study.

Previous randomized controlled trials have not supported moderate intensity exercise as an efficacious adjunct to smoking cessation treatments for women; however, compliance with exercise programs in these studies has been poor. The purpose of this pilot study was to estimate the effects of moderate intensity exercise on smoking cessation outcomes under optimal conditions for exercise program compliance. Sixty previously sedentary, healthy, female smokers were randomized to an 8-week program consisting of brief baseline smoking cessation counseling and the nicotine patch plus either 150 min/week of moderate intensity exercise or contact control. Participants attended a median of 86.4% and 95.5% of prescribed exercise/control sessions, respectively. There was a moderate, though statistically nonsignificant, effect of exercise at post-treatment for objectively verified 7-day point prevalence abstinence (48.3% vs. 23.3%; OR = 3.07, 95% CI: 0.89–11.07) and prolonged abstinence (34.5% vs. 20.0%; OR = 2.11, 95% CI: 0.56–8.32). Effects were attenuated when controlling for potential confounders, and after a 1-month, no-treatment period. The findings provide a preliminary indication that, given adequate compliance, moderate intensity exercise may enhance short-term smoking cessation outcomes for women; however, a larger trial is warranted. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

A rotating disk study of silver dissolution with thiourea in the presence of ferric sulfate

The rotating disk technique was used to study silver dissolution with thiourea as a function of sulfuric acid, ferric sulfate, and thiourea concentrations. The effect of many foreign ions (Mn, Cu, Co, Ca, Na,etc.) and various additives was also examined. The dissolution of silver was zero order with sulfuric acid, first order with ferric sulfate, and second order with thiourea. Among the foreign ions, copper had a dramatically negative effect. The strong oxidants such as hydrogen peroxide and manganese dioxide were also detrimental for silver dissolution. According to the temperature effect studied (5 °C to 35 °C), the activation energy was 22.6 kJ/ mole. Silver does not dissolve with thiourea in the absence of ferric ions. Sulfuric acid does not participate in the dissolution reaction. The most important parameter for silver dissolution is the ferric sulfate/thiourea ratio. In excess ferric sulfate, a solid silver-thiourea complex is formed, which precludes transfer of silver into solution. In excess thiourea, the free thiourea reacts with formed solid silver-thiourea complex, and silver goes into the solution, predominantly as the dimers of AgTU⁺ ₃ complexes. The solid silver-thiourea complex in question was characterized by various spectroscopic, microscopic, and chemical analysis techniques. According to chemical composition, it corresponds to Ag₂SO₂·3TUH₂O compound. Formerly Graduate Student, University of Idaho     

Thermodynamic features and an experimental study of the extraction of phosphorus from ferrophosphorus in the presence of iron silicides

The displacement of phosphorus from ferrophosphorus (24.89% P, 67.82% Fe) by FeSi₂ is studied by thermodynamic simulation. The melting of electrothermic ferrophosphorus with FS65 ferrosilicium with the extraction of gaseous phosphorus and the formation of a complex ferroalloy containing 42.8–44.3% Si, 50–52.4% Fe, 1.8–2.0% Mn, and 1.4–1.6% P is experimentally investigated.