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Dentin resorption mediated by odontoclasts in physiological root resorption of human deciduous teeth.

Extracted human deciduous teeth undergoing physiological root resorption were fixed with a mixture of formaldehyde and glutaraldehyde and processed for scanning (SEM) and analytical transmission electron microscopy, as well as acid trimetaphosphatase cytochemistry. The granulated tissues, which are rich in multinucleated odontoclasts and capillary vessels, formed various resorption lacunae on the resorbing dentin surfaces. SEM observations of dentin surfaces treated with sodium hypochlorite revealed two types of resorption lacunae: deep, round lacunae in which the peritubular matrix of dentinal tubules was strongly dissolved; and shallow, irregular lacunae with intact peritubular matrix. In trypsin-treated materials, the resorption surfaces were characterized by the presence of numerous collagen fibers in both the peritubular and intertubular matrices, suggesting demineralization of the surface dentin. Odontoclasts were characterized by the presence of abundant mitochondria, perinuclear stacks of Golgi membranes, various lysosomes, numerous endocytotic vacuoles, and a well-developed ruffled border against the resorption lacunae. Most endocytotic vacuoles were distributed in the cytoplasm between the ruffled border and the nuclei. In undemineralized ultrathin sections, the surface dentin of resorption lacunae consisted of collagen fibers and apatite crystals and had a lower packing density than those in unresorbed, deeper dentin. Many apatite crystals were demonstrated to be present in the extracellular channels of the ruffled border and in adjacent endocytotic vacuoles derived from it. Lysosomes located in the perinuclear cytoplasm of odontoclasts contained amorphous dense material and/or a small amount of crystals. An energy-dispersive x-ray microanalysis of apatite crystals in undemineralized sections indicated that the energy spectrum peaks of Ca and P detected from crystals in resorbing dentin were much lower than those in unresorbed dentin. Similarly, lower spectrum peaks of Ca and P were obtained from crystals found in the ruffled border and endocytotic vacuoles of odontoclasts. A slight trace Ca peak also was detected in the amorphous dense material in lysosomes of odontoclasts. The enzyme cytochemistry of lysosomal acid trimetaphosphatase indicated that odontoclasts had intense enzymatic activity in the Golgi membranes, endoplasmic reticulum cisternae, lysosomes, and endocytotic vacuoles. Dense reaction precipitates of enzymatic activity also were found along the dentin surfaces of resorption lacunae occupied by odontoclast ruffled borders.(ABSTRACT TRUNCATED AT 400 WORDS)

Acid Anhydride Hydrolases↗

Experimental hydroxyapatite synovial and articular cartilage calcification: light and electron microscopic studies.

Intraarticular calciphylaxis with cartilage and synovial calcification was produced in rabbits by oral administration of a single dose of dehydrotachysterol followed by an intraarticular injection of ferrous chloride. Synovial membrane apatite deposits were seen in the interstitium, on collagen fibers, and within cell vacuoles. In contrast, long-term administration of dehydrotachysterol alone induced only articular cartilage calcification. Such calcification was limited to the mid and deep zone of cartilage. Most calcium deposits were apatite-like crystals. Acute crystal-associated inflammation was not demonstrated in these specimens with crystals sequestered in synovial or cartilage tissue. Further studies on these models will examine the relation of apatite to joint disease.

Animals↗

Effects of ions in aqueous media on hydroxyapatite induction by silica gel and its relevance to bioactivity of bioactive glasses and glass-ceramics.

Hydroxyapatite induction by a synthesized pure silica hydrogel was examined in various simulated body fluids (SBFs) having different magnesium, calcium, and phosphate ion concentrations as well as pH values. The silica hydrogel generated biologically active apatite on its surface by taking up calcium and phosphorous ionic groups from a surrounding SBF that was prepared to emulate the human plasma in inorganic composition. The induction period for apatite nucleation on the surface of the silica was largely decreased with the addition of a small amount of the calcium or phosphate ions to the SBF and with an increase in pH, but increased with the addition of magnesium ion. Bioactivity of bioactive materials like Bioglass and glass-ceramic A-W was well interpreted in terms of the rate of apatite formation reflected in these results. Moreover, the results provide the basic knowledge for designing new bioactive materials.

Biocompatible Materials↗

In vitro behavior of albumin-loaded carbonate hydroxyapatite gel.

Hydroxyapatite (HA) powder, porous HA, plasma-sprayed HA, apatite cements, and sintered HA have all been investigated as delivery systems for compounds such as human growth hormone and vancomycin. However, many previous studies showed that the period of release was limited to 2-3 weeks. The concept of using a nanoporous matrix as a means of immobilizing proteins is well known but has largely been confined to silica-based systems. Carbonate hydroxyapatite (CHA) is more soluble in vivo than HA, and when formed as an aqueous precipitate, it is often formed as nanocrystals. This study investigated the release profiles of ovine albumin (OVA) from CHA gel stored in phosphate-buffered saline (PBS) and double distilled water (DDW) for times of up to 1 year. It was found that 7.9% OVA could be loaded onto apatitic gels by means of a purely aqueous process. This process provided a simple low-temperature method of protein adsorption on a high surface area apatitic matrix at physiological pH. The rate of short-term release of OVA was lower from CHA gels than from microcrystalline HA powder. However, the period of release from the CHA gel was short term and may have been associated with recrystallization of the gel. OVA loaded into CHA gel was found to remain undegraded in vitro at 37 degrees C for periods of up to 1 year.

Albumins↗

Reactivity of plasma-sprayed wollastonite coating in simulated body fluid.

We incubated wollastonite coatings prepared with plasma spraying in simulated body fluids for different periods to investigate the reactivity. The surface structures of wollastonite coatings immersed in simulated body fluids were analyzed with scanning electron microscopy, Auger electron spectroscopy, and transmission electron microscopy. The results showed a carbonate-containing hydroxyapatite layer formed on the surface of a plasma-sprayed wollastonite coating soaked in simulated body fluids, and a silica-rich layer appeared on the surface of the coating before the formation of the carbonate-containing hydroxyapatite layer. The formation mechanism of apatite on the coating surface could be explained in terms of the ionic exchange between H(+) within simulated body fluids and Ca(2+) in the coating, which resulted in an increase in the ionic activity product of the apatite in simulated body fluids and provided a specific surface with a lower interface energy to the formation of apatite nuclei.

Biocompatible Materials↗

Plasma sprayed coatings of hydroxylapatite.

The technique of plasma spraying has been applied to deposit a thin, dense layer of hydroxylapatite onto a titanium substrate. Bond strength of such apatite coatings with the substrate have been measured, as well as the (absence of) influence of the coating process on fatigue properties of the substrate. Animal studies showed similar histological reactions to apatite coatings as to (well documented) apatite bulk materials.

Animals↗

Physicochemical characterization of deposits associated with HA ceramics implanted in nonosseous sites.

Pellets of well-characterized microporous hydroxyapatite (HA) ceramic were implanted in hamsters in two nonosseous sites: (1) in the fatty tissue of the gingival crease, far from bony tissue and (2) in intraperitoneal sites. The implants in site 1 were placed directly in contact with tissues, cells, and extracellular fluids while the implants in site 2 were placed in special chambers made of plexiglass cylinders covered in both ends with millipore filters, preventing contact with tissues and cells, but not with extracellular fluids. The hamsters were sacrificed and the implants recovered after 8, 16, 30, 150, and 365 days. The pellets were characterized using x-ray diffraction, infrared absorption, thermogravimetry, scanning and transmission electron microscopy, and calcium and phosphate analyses before and after implantation. Physicochemical analyses of HA ceramic implants before and after implantation demonstrated the formation of new material which was significantly different from the HA ceramic in terms of the following: (a) morphology (size of shape) of crystals; (b) intimate association of the inorganic phase of the new material with an organic phase similar to inorganic/organic association in bone; (c) the inorganic phase of the new material is a CO3-apatite, similar to that of bone, while the HA in ceramic is CO3-free; (d) electron diffraction of apatite of new material is similar to that of bone apatite. This study also demonstrated that the new material associated with the HA ceramics implanted in two different nonosseous sites were identical in spite of the differences in their microenvironment (cellular and acellular).

Animals↗

Surface reactions of calcium phosphate ceramics to various solutions.

The surface reactions of calcium phosphate ceramics have been thought to play an important role in bonding with living bone. Hydroxyapatite (HA), tricalcium phosphate (TCP), and two kinds of apatite-containing glass ceramics were immersed in three types of solutions with different chemical constituents. The first solution was a physiological saline, the second contained phosphate (PO4), and the third was a balanced salt solution consisting of calcium (Ca), magnesium (Mg), and PO4. After serial incubation periods, changes in the solutions were assessed by measurement of total Ca, Mg, and PO4. The ceramic surfaces were studied using scanning electron spectroscopy, infrared reflection spectroscopy, and thin-film x-ray diffraction. The surface reactions of the ceramics were greatly affected by the chemical compositions of the surrounding media. In the complete solution with both Ca and PO4, a carbonated apatite layer was formed on the surfaces of HA, TCP, and the glass ceramics. In comparison to HA and TCP, the glass ceramics were characterized as Ca-releasing materials, the dissolved Ca creating an apatite layer on the surfaces in a few days, in conjunction with PO4 stock in the surrounding media. The immersion test with various solutions proved to be a simple and effective method of assessing surface conditions of ceramic materials.

Biocompatible Materials↗

Bone bonding mechanism of beta-tricalcium phosphate.

It has been proposed that the formation of a surface apatite layer in vivo on surface active ceramics is an essential condition for chemical bonding between ceramics and bone tissue. To clarify the difference in bone-bonding mechanisms between surface active ceramics and bioresorbable ceramics, two experiments were performed using plates of dense beta-tricalcium phosphate (beta-TCP). First, plates of beta-TCP were implanted subcutaneously in rats for 8 weeks. Surface change due to bioresorption was observed with scanning electron microscopy. Formation of the apatite layer on the surface was investigated using thin-film x-ray diffraction and Fourier transform infrared reflection spectroscopy. Second, plates of beta-TCP were implanted in tibiae of rabbits for 8 and 25 weeks and subjected to the detaching test to measure bone-bonding strength. beta-TCP bonded strongly to bone. Undecalcified sections of the interface of bone and beta-TCP were examined with SEM-EPMA. However, by physicochemical methods, no formation of surface apatite layer was observed. These results suggest that beta-TCP bonds to bone through microanchoring between bone and rough surface of resorbed beta-TCP.

Animals↗

Differences in ceramic-bone interface between surface-active ceramics and resorbable ceramics: a study by scanning and transmission electron microscopy.

The interface between bioactive ceramics and bone was studied by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The materials were apatite-wollastonite-containing glass ceramic (A-W.GC) as a representative surface-active ceramic, and calcite and beta-tricalcium phosphate (beta-TCP) as resorbable ceramics. Particles of these materials, ranging between about 100 microns and 300 microns in diameter, were implanted into rat tibiae, and specimens were prepared for observation at 8 weeks after implantation. Both SEM and TEM demonstrated that A-W.GC was bonded to bone through a thin Ca-P-rich layer consisting of fine apatite crystals apparently different from those of bone in shape, size, and orientation. Collagen fibers of the bone reached the surface of this layer, and chemical bonding between A-W.GC and the bone was speculated. Calcite and beta-TCP, on the other hand, made direct contact with the bone, and no apatite layer was present at the interface. The surfaces of the implants became rough due to degradation, and bone grew into the finest surface irregularities. However, we were unable to demonstrate any continuity of crystals between the resorbable implants and bone by high-resolution TEM. Accordingly, the bonding strength was considered to be mainly attributable to mechanical interlocking.

Animals↗

The effect of calcium phosphate ceramic composition and structure on in vitro behavior. II. Precipitation.

The formation of a biologically equivalent carbonate-containing apatite on the surface of synthetic calcium phosphate ceramics (CPC) may be an important step leading to bonding with bone. Reactions of several single phases CPCs upon immersion into a simulated physiologic solution (SPS) with an electrolyte composition of human plasma were determined. The CPCs covered a wide range of solution stabilities from low-soluble hydroxyapatites (HA) to metastable tricalcium phosphates (TCP) and tetracalcium phosphate (TTCP). Changes in chemical compositions of SPS and infrared spectral features after CPC immersion were analyzed. New phase formation was observed on all the CPCs. However, kinetics, compositions, and structures of the new phases were significantly different. The studied CPCs can be characterized by the time to new phase formation in vitro; the minimum time for measurable precipitate formation was found to increase in the order: not-well-crystallized HAs < well-crystallized HAs < alpha-TCP, TTCP < beta-TCP. Among the CPCs only not-well-crystallized HAs led to immediate new phase formation. The metastable CPCs, beta-TCP, alpha-TCP, and TTCP required an induction time during which dissolution occurred. beta-TCP showed the longest induction time and the lowest lattice ion uptake rate of all the CPCs tested. Only the not-well-crystallized HAs elicited immediate formation of carbonated HA. The well-crystallized HAs and beta-TCP did not elicit carbonated apatite formation within the time frame of the experiment. Instead, intermediate phases were formed. On alpha-TCP amorphous calcium phosphate (ACP) with a relatively low carbonate content was formed. TTCP was found to transform extensively to poorly crystallized carbonated apatite after 2 days of immersion.

Biocompatible Materials↗

Physicochemical properties of calcific deposits isolated from porcine bioprosthetic heart valves removed from patients following 2-13 years function.

The purpose of this study was to characterize the physicochemical properties of calcific deposits that cause the failure of tissue-derived heart valve bioprostheses. This was done in an effort to understand the mechanism of pathologic biomineralization in the cardiovascular system and potentially prevent deterioration of bioprostheses. Calcific deposits taken from 10 failed bioprosthetic valves that had been implanted in patients for 2-13 years were characterized by chemical analysis, x-ray diffraction, FTIR spectroscopy, scanning electron microscopy, polarized light microscopy, and solubility measurements. The combined results identified the biomineral as an apatitic calcium phosphate salt with substantial incorporation of sodium, magnesium and carbonate. The average Ca/PO4 ratio for this "young" pathologic biomineral was approximately 1.3, considerably lower than approximately 1.7 found in mature atherosclerotic plaque biomineral and mature skeletal biomineral, both of which approximate hydroxyapatite in composition. Deproteinated calcific deposits from bioprostheses had thermodynamic solubilities comparable to those of both atherosclerotic plaque, typical pathologic biomineral and hydrolyzed octacalcium phosphate (OCP, Ca4H(PO4)3 x 2.5 H2O), a proposed precursor phase to biomineral apatite. This later finding, together with chemical composition and structural details of the bioprosthetic deposits themselves, supports a mechanism of cardiovascular calcification in which OCP plays a crucial role in the formation of the final apatitic phase. This suggests an approach toward prevention of bioprosthetic tissue calcification through control of the formation of the kinetically favored OCP precursor and/or its transformation into bioapatite.

Bioprosthesis↗

Dissolution properties of calcium phosphate granules with different compositions in simulated body fluid.

Calcium phosphate granules were produced through the calcination of three hydroxyapatites (HAs) at 1150 degrees C: nearly-stoichiometric (NS-HA), calcium-deficient (CD-HA), and carbonated (Carb-HA). The characterization using scanning electron microscope (SEM), X-ray diffraction (XRD), and Fourier-transformed infrared spectroscopy (FT-IR) showed that those granules exhibit strong differences in chemical composition, surface texture, and dissolution behavior. Sample dissolution in a simulated body fluid (SBF) and precipitation of a calcium phosphate layer on the granule surface were followed up for 7 days by chemical analysis of calcium content in SBF. After 80-min reaction, precipitation of new calcium phosphate phases predominates over the dissolution of original granules. Comparison between SEM images and XRD/FT-IR analysis revealed that the composition of the calcium phosphate layer and its precipitation rate depend on the HA composition and its heat treatment. Calcined carbonated apatite induces the precipitation of an octacalcium phosphate (OPC) layer, whereas a carbonated apatite layer is formed from calcined-deficient HA. The calcined nearly-stoichiometric HA exhibits low efficiency in producing carbonated apatite layer.

Biocompatible Materials↗

In vitro osteoblast response to anodized titanium and anodized titanium followed by hydrothermal treatment.

In this study, Titanium (Ti) surfaces were modified using anodization. The electrolyte used for anodization was a mixture of calcium glycerophosphate and calcium acetate. The anodized surfaces were divided into three groups. Hydrothermal treatments were performed on two of the anodized groups for either 2 or 4 h. In vitro osteoblast response to anodized oxide and the hydrothermal treated oxide after anodization was evaluated in this study. Calcium and phosphorus ions were deposited on the Ti oxide during anodization. Anodized surfaces following a 4-h hydrothermal treatment were observed to promote the growth apatite-like crystals as compared with anodized surfaces after a 2-h hydrothermal treatment. Cellular function and onset of mineralization, as indicated by protein production and osteocalcin production, respectively, also were observed as enhanced on hydrothermal-treated surfaces. It was thus concluded from this study that calcium phosphate and apatite-like crystals could be deposited on Ti surfaces using anodization and a combination of anodization and hydrothermal treatment. It was also concluded that the phenotypic expression of osteoblast was enhanced by the presence of calcium phosphate or apatite-like crystals on anodized or hydrothermally treated Ti surfaces.

Alkaline Phosphatase↗

Use of sol-gel-derived titania coating for direct soft tissue attachment.

A firm bond between an implant and the surrounding soft tissue is important for the performance of many medical devices (e.g., stents, canyls, and dental implants). In this study, the performance of nonresorbable and reactive sol-gel-derived nano-porous titania (TiO(2)) coatings in a soft tissue environment was investigated. A direct attachment between the soft tissue and the sol-gel-derived titania coatings was found in vivo after 2 days of implantation, whereas the titanium control implants showed no evidence of soft tissue attachment. The coated implants were in immediate contact with the connective tissue, whereas the titanium controls formed a gap and a fibrous capsule on the implant-tissue interface. The good soft tissue attachment of titania coatings may result from their ability to initiate calcium phosphate nucleation and growth on their surfaces (although the formation of poorly crystalline bonelike apatite does not occur). Thus, the formation of a bonelike CaP layer is not crucial for their integration in soft tissue. The formation of bonelike apatite was hindered by the adsorption of proteins onto the initially formed amorphous calcium phosphate growth centers, thus preventing the dissolution/reprecipitation processes required for the formation of poorly crystalline bonelike apatite. These findings might open novel application areas for sol-gel-derived titania-based coatings.

Adhesiveness↗

Hydroxyapatite growth on anodic TiO2 nanotubes.

In the present work, we study the growth of hydroxyapatite formation on different TiO(2) nanotube layers. The nanotube layers were fabricated by electrochemical anodization of titanium in fluoride-containing electrolytes. To study various nanotube lengths, layers with an individual tube diameter of 100 nm were grown to a thickness of approximately 2 mum or 500 nm. The ability to form apatite on the nanotube layers was examined by immersion tests combined with SEM, XRD and FT-IR investigations. For reference, experiments were also carried out on compact anodic TiO(2) layers. The results clearly show that the presence of the nanotubes on a titanium surface enhances the apatite formation and that the 2-mum thick nanotube layer triggers deposition faster than the thinner layers. Tubes annealed to anatase, or a mixture of anatase and rutile are clearly more efficient in promoting apatite formation than the tubes in their "as-formed" amorphous state.

Coated Materials, Biocompatible↗

In vitro bioactivity and osteoblast response of porous NiTi synthesized by SHS using nanocrystalline Ni-Ti reaction agent.

Porous NiTi with an average porosity of 55 vol % and a general pore size of 100-600 microm was synthesized by self-propagating high temperature synthesis (SHS) with the addition of mechanically alloyed nanocrystalline Ni-Ti as the reaction agent. The SHS of porous NiTi using elemental powders was also performed for comparison. To enhance the bioactivity of the metal surface, porous NiTi synthesized by nanocrystalline Ni-Ti was subjected to chemical treatment to form a layer of TiO(2) coating. The porous NiTi with TiO(2) coating was subsequently immersed in a simulated body fluid (SBF) to investigate its apatite forming ability. The effects of the addition of nanocrystalline Ni-Ti as reaction agent and the application of apatite coating on osteoblastic behavior were studied in primary cultures of human osteoblast cells. Results showed that the main phases in porous NiTi synthesized by elemental powders were NiTi, Ti(2)Ni, and unreacted free Ni. By using nanocrystalline Ni-Ti as reaction agent, the secondary intermetallic phase of Ti(2)Ni was significantly reduced and the free Ni was eliminated. TiO(2) coating with anatase phase was formed on the surface of porous NiTi after the chemical treatment. A layer consisting of nanocrystalline carbonate-containing apatite was formed on the surface of TiO(2) coating after soaking in SBF. The preliminary cell culture studies showed that the porous NiTi synthesized with the addition of nanocrystalline Ni-Ti attracted marked attachment and proliferation of the osteoblast cells. This gives the evidence of the potential biomedical applications of the porous NiTi.

Biocompatible Materials↗

Calcium carbonate-calcium phosphate mixed cement compositions for bone reconstruction.

The feasibility of making calcium carbonate-calcium phosphate (CaCO(3)-CaP) mixed cements, comprising at least 40% (w/w) CaCO(3) in the dry powder ingredients, has been demonstrated. Several original cement compositions were obtained by mixing metastable crystalline CaCO(3) phases with metastable amorphous or crystalline CaP powders in aqueous medium. The cements set within at most 1 h at 37 degrees C in atmosphere saturated with water. The hardened cement is microporous and exhibits weak compressive strength. The setting reaction appeared to be essentially related to the formation of a highly carbonated nanocrystalline apatite phase by reaction of the metastable CaP phase with part or almost all of the metastable CaCO(3) phase. The recrystallization of metastable CaP varieties led to a final cement consisting of a highly carbonated poorly crystalline apatite analogous to bone mineral associated with various amounts of vaterite and/or aragonite. The presence of controlled amounts of CaCO(3) with a higher solubility than that of the apatite formed in the well-developed CaP cements might be of interest to increase resorption rates in biomedical cement and favors its replacement by bone tissue. Cytotoxicity testing revealed excellent cytocompatibility of CaCO(3)-CaP mixed cement compositions.

Biocompatible Materials↗