Collagen-hydroxyapatite films: piezoelectric properties

In this paper we report a study of the physicochemical, dielectric and piezoelectric properties of anionic collagen and collagen-hydroxyapatite (HA) composites, considering the development of new biomaterials which have potential applications in support for cellular growth and in systems for bone regeneration. The piezoelectric strain tensor element d(14), the elastic constant s(55) and the dielectric permittivity 8(11), were measured for the anionic collagen and collagen-HA films. The thermal analysis shows that the denaturation endotherm is at 59.47 degreesC for the collagen sample. The collagen-HA composite film shows two transitions, at 48.9 and 80.65 degreesC. The X-ray diffraction pattern of the collagen film shows a broad band characteristic of an amorphous material. The main peaks associated to the crystalline HA is present in the sample of collagen-HA. In the collagen-HA composite, one can also notice the presence of other peaks with low intensities which is an indication of the formation of other crystalline phases of apatite. The scanning electron photomicrograph of anionic collagen membranes shows very thin bundles of collagen. The scanning electron photomicrography of collagen-HA film also show deposits of hydroxyapatite on the collagen fibers forming larger bundles and suggesting that a collagenous structure of reconstituted collagen fibers could act as nucleators for the formation of apatite crystal similar to those of bone. The piezoelectric strain tensor element d(14) was measured for the anionic collagen, with a value of 0.062 pC N-1, which is in good agreement compared with values reported in the literature obtained with other techniques. For the collagen-HA composite membranes, a slight decrease of the value of the piezoelectricity (0.041 pC N-1) was observed. The anionic collagen membranes present the highest density, dielectric permittivity and lowest frequency constant f.L. (C) 2001 Elsevier B.V. B.V. All rights reserved.

Direct determination of phosphite in fertilizers by flow-injection amperometry

A sensor based on graphite electrode modified with palladium-platinum-palladium film is proposed for phosphite determination by flow-injection amperometry. The modified electrode was prepared by a sequential cathodic deposition of Pd, Pt and Pd on a graphite electrode from 0.5% m/v PdCl2 + 28% m/v NH4OH and 2% m/v H2PtCl6 + 10% v/v H2SO4 solutions. After suitable conditioning, the electrode showed catalytic activity for phosphite oxidation when 0. 15 V was applied. The proposed system handles approximately 50 samples per hour (0.0.1 - 0.05 mol L-1 Na-2 HPO3; R-2 = 0.9997), consuming ca. 70 mu L of sample per determination. The limit of detection and amperometric sensibility were 5 X 10(-4) mol L-1 and 1.5 mA L mol(-1), respectively. The proposed method was applied to analysis of fertilizer samples without pre-treatment. Results are in agreement with those obtained by spectrophotometry and titrimetry at 95% confidence level and good recoveries (96-109%) of spiked samples were found. Relative standard deviation (n=12) of a 0.01 mol L-1 Na2HPO3 sample was 2%. The useful lifetime of modified electrode was around 220 determinations. For routine purposes it means that this electrode can be continuously used for 5 hours.

Collagen type VI is a component of the extracellular matrix microfibril network of the prostatic stroma

Stroma-epithelium relationships are of great relevance in prostatic morphogenesis and physiology, However, little knowledge exists about either stromal cells or extracellular matrix composition and arrangement in this system, Ultrastructural analysis revealed the existence of a microfibrillar system which occupies large areas of the rat prostatic stroma, In this work, we have applied immunocytochemistry and an ATP treatment for the ultrastructural identification of collagen type VI microfibrils, aiming at examining its participation in the prostatic microfibrillar network. Immunocytochemistry was also extended to a human case of prostatic nodular hyperplasia, Both methods succeeded in identifying collagen type VI in the rat ventral prostate, Collagen type VI is evenly distributed throughout the stroma but mainly associated with the basal lamina, collagen fibrils, and around the stromal cells, the use of ATP treatment allowed for the discrimination between collagen type VI and elastin-associated microfibrils, and demonstrated that these two classes of microfibrils establish an extended, mixed, and open network. The same aspects of association with the basal lamina, with stromal cells (particularly with smooth muscle cells), and with fibrillar components of the stroma were observed in the human tissue, We suggest that the collagen type VI and elastin-associated microfibril system may be involved in the control of some aspects of cellular behavior and may also play a structural role, maintaining the organ integrity after the deformations occurring under smooth muscle contraction.

INTRATRACHEAL INJECTION OF NICKEL CHLORIDE AND COPPER-ZINC SUPEROXIDE-DISMUTASE ACTIVITY IN LUNG OF RATS

Superoxide radical (O2-) is a free radical that may be involved in various toxic processes. Cu-Zn superoxide dismutase catalyses the dismutation of the superoxide free radical and protects cells from oxidative damage, and it has been used clinically. The concentration of Ni2+ and Cu-Zn superoxide dismutase activity were measured in lungs of rats at time intervals of 5, 12, 19, 26, 33, and 40 days following an intratracheal injection of 127 nmol of NiCl2. Nickel chloride increased nickel content and resulted in a significant increase of Cu-Zn superoxide dismutase activity in lungs. This elevation of Cu-Zn superoxide dismutase activity was highest on the 12th day (approximately threefold) and is at levels comparable to controls rats on day 40 onwards. Since Cu-Zn superoxide dismutase activity was increased in lung throughout our experimental period without corresponding increases of Cu2+ and Zn2+, we speculate that the elevation of Cu-Zn superoxide dismutase activity might be due to an increased half-life of the enzyme, induced by nickel.

Flow injection AAS determination of Cd, Cu, and Pb at trace levels in wine using solid phase extraction

A comparative study is reported between C-18 bonded silica gel and powdered polyethylene (PE) as sorbent for Cd, Cu, and Pb determination using ammonium diethyldithiophosphate (ADTP) as the complexing agent in a flow injection system. The complexes were formed in 0.14 mol L-1 HNO3 and processed in a simple flow system comprising a peristaltic pump, a manual injector-commutator, and a sorbent-packed minicolumn. Ethanol was selected as the eluent and analytes in the eluate were determined by flame atomic absorption spectrometry. The optimum concentration of the complexing agent was 0.1% (m/v) ADTP for Cu and Pb determination using either C-18 or PE, and 0.25% (m/v) ADTP for Cd determination using PE. The sample loading flow rates were 5.0, 3.6, and 3.0 mL min(-1) for Cu, Pb, and Cd, respectively. The best elution flow rate was 6.5 mL min(-1). For a 60-sec preconcentration time, the sampling rate was 40 h(-1) and the enrichment factors of 33, 36, and 11 times (C-18) or 18, 22, and 23 times (PE) were obtained for Cu, Pb, and Cd, respectively. The limits of detection (LOD) were 1.6 mug L-1 Cu, 11 mug L-1 Pb, and 2.0 mug L-1 Cd using C-18 or 2.9 mug L-1 Cu, 19 mug L-1 Pb, and 1.0 mug L-1 Cd using PE, respectively. The relative standard deviations (n = 12) were typically <2%, <2%, and <6% for Cd, Cu, and Pb, respectively. The recoveries of Cd, Cu, and Pb added to wine samples varied from 96-99%, 97-102%, and 90-99%, respectively, using C-18 or PE. Accuracy was checked for Cd, Cu, and Pb determination in six wine samples digested by block digestor and open-vessel microwave-assisted digestion systems. The results revealed that C-18 was more efficient for Cu and Pb determination, while PE was the best sorbent for Cd.

Evaluation of sample treatment for the simultaneous flow injection hydride generation and determination of As, Bi, Sb and Se by GRAS

A new method was developed for the simultaneous determination of As, Bi, Sb, and Se by flow injection hydride generation graphite furnace atomic absorption spectrometry. An alternative two-step sample treatment procedure was used. The sample was heated (80degreesC) for 10 min in 6 M HCl to reduce Se(VI) to Se(IV), followed by the addition of 1% (m/v) thiourea solution to reduce arsenic and antimony from the pentavalent to the trivalent states.With this procedure, all analytes were converted to their most favorable and sensitive oxidation states to generate the corresponding hydrides. The pre-treated sample solution was then processed in the flow system for in situ trapping and atomization in a graphite tube coated with iridium. The impermanent modifier remained stable up to 300 firings and new coating out significant were possible wit changes in the analytical performance.The accuracy was checked for As, Bi, Sb, and Se determination in water standard reference materials NIST 1640 and 1643d and the results were in agreement with the certified values at a 95% confidence level. Good recoveries (94-104%.) of spiked mineral waters and synthetic As(V), Sb(Ill), mixtures of As(Ill), Sb(V), Se(VI), and Se(IV) were also found. Calculated characteristic masses were 32 mug As, 79 mug Bi, 35 mug Sb, and 130 pg Se, and the corresponding limits of detection were 0.06, 0.16, 0.19, and 0.59 mug L-1, respectively. The repeatability for a typical solution containing 5 mug L-1 As, Bi, Sb, and Se was in the 1-3% range.

Spectrophotometric detection of arsenic using flow-injection hydride generation following sorbent extraction preconcentration

An automated system with a C-18 bonded silica gel packed minicolumn is proposed for spectrophotometric detection of arsenic using flow-injection hydride generation following sorbent extraction preconcentration. Complexes formed between arsenic(III) and ammonium diethyl dithiophosphate (ADDP) are retained on a C-18 sorbent. The eluted As-DDP complexes are merged with a 1.5% (w/v) NaBH4 and the resulting solution is thereafter injected into the hydride generator/gas-liquid separator. The arsine generated is carried out by a stream of N-2 and trapped in an alkaline iodine solution in which the analyte is determined by the arsenomolybdenum blue method. With preconcentration time of 120 s, calibration in the 5.00-50.0 mu g As l(-1) range and sampling rate of about 20 samples h(-1) are achieved, corresponding to 36 mg ADDP plus 36 mg ammonium heptamolybdate plus 7 mg hydrazine sulfate plus 0.7 mg stannous chloride and about 7 mi sample consumed per determination. The detection limit is 0.06 mu g l(-1) and the relative standard deviation (n = 12) for a typical 17.0 mu g As l(-1) sample is ca. 6%. The accuracy was checked for arsenic determination in plant materials from the NIST (1572 citrus leaves; 1573 tomato leaves) and the results were in agreement with the certified values at 95% confidence level. Good recoveries (94-104%) of spiked tap waters, sugars and synthetic mixtures of trivalent and pentavalent arsenic were also found. (C) 1999 Elsevier B.V. B.V. All rights reserved.

Implants of polyanionic collagen matrix in bone defects of ovariectomized rats

In recent years, there has been a great interest in the development of biomaterials that could be used in the repair of bone defects. Collagen matrix (CM) has the advantage that it can be modified chemically to improve its mechanical properties. The aim of the present study was to evaluate the effect of three-dimensional membranes of native or anionic (submitted to alkaline treatment for 48 or 96 h) collagen matrix on the consolidation of osteoporosis bone fractures resulting from the gonadal hormone alterations caused by ovariectomy in rats subjected to hormone replacement therapy. The animals received the implants 4 months after ovariectomy and were sacrificed 8 weeks after implantation of the membranes into 4-mm wide bone defects created in the distal third of the femur with a surgical bur. Macroscopic analysis revealed the absence of pathological alterations in the implanted areas, suggesting that the material was biocompatible. Microscopic analysis showed a lower amount of bone ingrowth in the areas receiving the native membrane compared to the bone defects filled with the anionic membranes. In ovariectomized animals receiving anionic membranes, a delay in bone regeneration was observed mainly in animals not subjected to hormone replacement therapy. We conclude that anionic membranes treated with alkaline solution for 48 and 96 h presented better results in terms of bone ingrowth.

Extracellular matrix of porcine pericardium: Biochemistry and collagen architecture

Pericardial tissue has been used to construct bioprostheses employed in the repair of different kinds of injuries, mostly cardiac. However, calcification and mechanical failure have been the main causes of the limited durability of cardiac bioprostheses constructed with bovine pericardium. In the course of this work, a study was conducted on porcine fibrous pericardium, its microscopic structure and biochemical nature. The general morphology and architecture of collagen were studied under conventional light and polarized light microscopy. The biochemical study of the pericardial matrix was conducted according to the following procedures: swelling test, hydroxyproline and collagen dosage, quantification of amino acids in soluble collagen, component extraction of the extracellular matrix of the right and left ventral regions of pericardium with different molarities of guanidine chloride, protein and glycosaminoglycan (GAG) dosage, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and total GAG analysis. Microscopic analysis showed collagen fibers arranged in multidirectionally oriented layers forming a closely knit web, with a larger number of fibers obliquely oriented, initiating at the lower central region toward the upper left lateral relative to the heart. No qualitative differences were found between proteins extracted from the right and left regions. Likewise, no differences were found between fresh and frozen material. Protein dosages from left frontal and right frontal pericardium regions showed no significant differences. The quantities of extracted GAGs were too small for detection by the method used. Enzymatic digestion and electrophoretic analysis showed that the GAG found is possibly dermatan sulfate. The proteoglycan showed a running standard very similar to the small proteoglycan decorin.

Collagen is reduced and disrupted in human aneurysms and dissections of ascending aorta

In ascending aorta aneurysms, there is an enlargement of the whole vessel, whereas aortic dissections (ADs) are characterized by the cleavage of the wall into 2 sheets at the external half. We searched if alterations in collagen could be related to these diseases. Sections of aortas from 14 case patients with acute dissections, 10 case patients with aneurysms, and 9 control subjects were stained with picrosirius. Slides were analyzed under polarized microscopy to evaluate the structure of collagen fibers. The proportion of collagen was calculated in each half of the medial layer by color detection in a computerized image analysis system. Collagen appearance under polarized light was consistent with collagenolysis. The mean collagen proportions at the inner and outer halves, respectively, were 0.50 +/- 0.13 and 0.40 +/- 0.08 in the control group, 0.20 +/- 0.10 and 0.18 +/- 0.12 in the AD group, and 0.33 +/- 0.12 and 0.19 +/- 0.12 in the aneurysm group. The AD (P < .01) and control (P = .04) groups had less collagen at the external half, no difference was found in the aneurysm group (P = .71). In both halves, there was less collagen in the case patients than in the control subjects (all P < .01), but at the internal half, the decrease was significantly greater in the case patients with aneurysms than in those with dissections (P = .03; at the external half, P = .99). Aortic dissections and aneurysms show a decrease in collagen content that could be related to a weakness of the wall underlying the diseases, but the locations of the decrease differ: in dissections, it is situated mostly at the external portion of the media (site of cleavage), whereas in aneurysms, it is more diffuse, consistent with the global enlargement. (c) 2008 Elsevier B.V. All rights reserved.

Densitometry, radiography, and histological assessment of collagen as methods to evaluate femoral bones in an experimental model of osteoporosis

All methods to detect experimental loss of bone present technique limitations. The sensitivities of image and histological analyses to detect the effects of teriparatide in rats with bone loss after ovariectomy were evaluated. All methods were qualitatively valid.The standardization of methods to assess bone loss after ovariectomy is crucial to establish the degree of experimental osteoporosis. In general, methods per image or histological techniques are used. To validate these two ways to determine the degree of bone loss in ovariectomized rats, we evaluated the sensitivities of bone densitometry, conventional radiography, and histological analysis of the area occupied by collagen, detecting the effects of teriparatide treatment in the femur of ovariectomized rats with bone loss.Wistar rats were divided into three groups: a control group, in which the animals were only subjected to laparotomy; an ovariectomized group, in which bilateral removal of the ovaries was performed; and an ovariectomized + teriparatide group, in which bilateral removal of the ovaries was performed, and the animals were treated with 3 mu g/100 g/day of teriparatide. Three months following the ovariectomy, bone densitometry, radiographic densitometry, and histological analysis of the area occupied by collagen fibers were carried out in the femur diaphysis.The bone densitometry revealed 11.2% reduction in femur density; in the conventional radiography, the loss of bone mass was 14.5%, and with the histological analysis, a 40.9% reduction in the area occupied by collagen was detected in the femur diaphysis.In conclusion, histological analysis could not be quantitatively compared with the methods of bone densitometry and conventional radiography; however, all of these methods were qualitatively valid for assessing the loss of bone stemming from ovariectomy and the therapeutic effect of teriparatide in the prevention of osteoporosis.