Calcified Hard Tissue Research Articles

Histological evaluation of hard tissue formation after direct pulp capping with a fast-setting mineral trioxide aggregate (RetroMTA) in humans.

To histologically evaluate the morphology of the newly formed mineralized tissue and of the adjacent cells, in intact human teeth subjected to mechanical pulp exposure and capping with a fast-setting mineral trioxide aggregate (RetroMTA). Seven caries-free third molars from three adults were subjected to pulp exposure, direct capping with RetroMTA, and restoration with a composite resin. Seven months later, the teeth were clinically and radiographically evaluated, extracted, and subjected to histological processing and evaluation. All teeth were clinically and radiographically inconspicuous and showed no presence of severe inflammatory reactions. Bacteria were absent in all teeth. All cases exhibited some degree of mineralized tissue in the area of exposure to varying extent. This newly formed mineralized tissue was mostly atubular and did not display the features of regular dentine in any of the cases. No cells exhibiting the features of odontoblasts or odontoblast-like cells were observed. Instead, the cells exhibited a flat or cuboidal shape, resembling fibroblasts. When the exposed pulps were directly capped with RetroMTA, the new calcified hard tissue was not "regular dentine," and did not seem to be the product of genuine odontoblast differentiation. These results suggest that the formation of calcified tissues after direct pulp capping with RetroMTA may be more appropriately regarded as a reparative process than as a genuine regeneration response. This is the first histological study on humans showing that regular dentine was not regenerated when a bioactive pulp-capping material (RetroMTA) was placed over exposed pulp tissue. NCT03631511.

Osseous choristoma of the tongue: A case report with dermoscopic study.

Osseous choristomas are rare benign lesions characterized by ectopic bone formation in the soft tissue of the head and neck region. Dermoscopy visualizes the morphological characteristics that are not observable by the naked eye, and may be used for the evaluation of calcification under the skin. The present study reports a case of an osseous choristoma arising in the tongue, and reveals the dermoscopic features of osseous choristoma from a surgical specimen. A 7-year-old boy was referred to the Department of Dentistry and Oral Surgery, with an asymptomatic pedunculated mass in the tongue. The lesion was removed completely with the adjacent normal tissue under general anesthesia. Dermoscopy of the surgical specimen revealed the hypovascular and homogeneous pattern of the lesion with round extruded whitish material. Based on dermoscopic findings, the presence of calcified hard tissue in the submucosa was verified by the dermatologist. Radiographic examination of the surgical specimen revealed the lesion contained a radiopaque trabeculated mass. Histologically, the lesion contained an osseous tissue, and the pathological diagnosis of osseous choristoma was made following consideration of the ectopic bony tissues that were localized away from the maxillo-mandibular bone. The postoperative course was uneventful with no signs of recurrence during the 36 months following surgery. To the best of the author's knowledge, this is the first report of evaluation of osseous choristoma using dermoscopy. The observation indicates the usefulness of dermoscopy for the diagnosis of oral ossified lesion in oral soft tissue.

Sinus Floor Augmentation Comparing an In Situ Hardening Biphasic Calcium Phosphate (Hydroxyapatite/β-Tricalcium Phosphate) Bone Graft Substitute with a Particulate Biphasic Calcium Phosphate (Hydroxyapatite/β-Tricalcium Phosphate) Bone Graft Substitute: An Experimental Study in Sheep.

The aim of the present split-mouth study in sheep was to assess the influence of in situ hardening properties of a biphasic calcium phosphate (BCP) bone graft substitute (BGS) (ratio hydroxyapatite/β-tricalcium phosphate = 60/40) compared with a particulate BGS with the same biphasic core-granule composition without in situ hardening properties on sinus floor augmentation. Therefore, bilateral sinus floor augmentation was performed in eight sheep. Poly(lactide-co-glycolide) (PLGA)-coated, in situ hardening biphasic BGS (PLGA-NMP [N-Methyl-2-pyrrolidone]-BCP) was placed at the test site, and a particulate biphasic BGS without PLGA coating (BCP) was used for the contralateral site as a control. Animals were sacrificed after 21 weeks. Sinus augmentation sites were analyzed histologically. The volume was analyzed by computed tomography. Histomorphometric parameters were assessed for the 12 and 21 weeks' time points. Slopes of new bone formation over time were compared with a linear growth regression model. Bone formation after 12 and 21 weeks of healing was 8.94% (±3.74) and 19.82% (±6.29) for PLGA-NMP-BCP and 7.00% (±2.58) and 14.38% (±4.51) for BCP, respectively. The bone growth rate for PLGA-NMP-BCP was higher than the growth rate for BCP (probability 97.5%). The total fraction of calcified hard tissue (% bone fraction + % biomaterial) was around 46% for both tested biomaterials, 21 weeks after sinus floor augmentation. The in situ hardening BGS (PLGA-NMP-BCP) performed better than the particulate material (BCP) in terms of bone formation rate. The in situ hardening properties of the PLGA-NMP-BCP material mediated by the PLGA coating and NMP solution as plasticizer had no negative influence on the bone formation.

From molecules to macrostructures: recent development of bioinspired hard tissue repair.

Enamel, dentin and bone are calcified hard tissues in the human body that play significant roles in food mastication and movement support. Generally, hard tissues lack the ability of self-repair, except for the regeneration ability of bone for small-scale defects. Fabrication of man-made repair materials is therefore highly demanded. In this review, following a bioinspired strategy, we describe the composition and multiscale structures of different hard tissues, and highlight the key points for the reconstruction of hard tissues. Finally, bioinspired tissue repair techniques ranging from molecule-induced mineralization, to microscale assembly to macroscaffold fabrication are summarised.

Surface chemistry of biological apatites in mineralized tissues (on the example of spongy bone tissue).

Background. It is well known that poorly crystallized carbonate apatite is the main mineral component of the normal calcified hard tissues of human skeleton. However, some attributes and characteristics of bioapatites in physiologically mineralized tissue remain undefined. It is thought that structural imperfections and surface properties of the bioapatite nanocrystals govern the crystal chemistry and therefore should be crucial factors in formation, resorption and remodeling behavior of the biominerals. The biocrystal surface plays an exceptional role as a boundary between mineral and organic components and location of the main processes. The correlations between structural defects and content of labile ions on the crystal surface may reflect some ultrastructural features of bioapatite and ability to ion exchange. Objective. The aim of the research was to study the crystal-chemical characteristics of bio-related calcium apatites. Methods. In our research we used pyrolysis and subsequent ultrasonication in aqueous medium. Because apatite solubility is minor at normal conditions, the free ions can be removed in aqueous solution, keeping unaffected apatite structure, and then their concentration can be determined by atomic spectroscopy (AS). Results. 60-70 % of Mg present in bone seems to be incorporated in bioapatite structure substituting Ca, while the remaining Mg is located on the bone mineral crystal surface where it is easily available and could be removed by ultrasonic treatment. Also we got evidence that Mg and Na in bone apatite can be both in structurally bounded (substituting calcium in lattice) and in labile state (localizing on the crystal surface), while K is not able to join the apatite structure in significant amount or to be chemically bounded to it.

Fluoride Exposure May Accelerate the Osteoporotic Change in Postmenopausal Women: Animal Model of Fluoride-induced Osteoporosis

Carbonic anhydrase is a key enzyme for initiating the crystal nucleation, seen as “the central dark line” in the crystal structure in calcified hard tissues such as tooth enamel, dentin and bone. Both estrogen deficiency and fluoride exposure adversely affected the synthesis of this enzyme in the calcifying hard tissues. This led to the notion that fluoride exposure might increase the risk of developing osteoporosis in postmenopausal women. Using ovariectomized rats, which represent an estrogen (Es)-deficient state, as an animal model of postmenopausal women, we examined the causal relationship between fluoride (F) exposure and risk of developing osteoporosis. Two groups of rats, an Es-deficient group and a non-Es-deficient group, were administered free drinking water containing F ions (1.0 mg/L). Two other groups, an Es-deficient group and a control-group, were administered tap water. Soft X-ray radiography demonstrated a significant increase of radiolucent areas in the calvaria of the combined Esdeficient plus F group compared to that in the other experimental groups. Electron microscopy revealed an increase of amorphous minerals in the radiolucent areas. Light microscopy demonstrated that combined effects evidently of Es-deficiency and administration of F caused deterioration of the rat tibia with a coarse pattern of trabecular architecture, suggesting that a decline in bone formation might be the primary cause of osteoporosis. Consequently, F exposure might accelerate osteoporotic changes in postmenopausal women even at a low dose.

Electrochemical removal of metallic implants from Technovit 9100 New embedded hard and soft tissues prior to histological sectioning

Solid metallic implants in soft or hard tissues are serious challenges for histological processing. However, metallic implants are more frequently used in e.g. cardiovascular or orthopaedic therapies. Before clinical use, these devices need to be tested thoroughly in a biological environment and histological analysis of their biocompatibility is a major requirement. To allow the histological analysis of metallic implants in tissues especially in calcified hard tissues, we describe a method for embedding these tissues in the resin Technovit 9100 New and removing the metallic implants by electrochemical dissolution. With the combination of these two processes, we are able to achieve 5μm thick sections from soft or hard tissues with a superior preservation of tissue architecture and especially the implant-tissue interface. These sections can be stained by classical stainings, immunohistochemical and enzymehistochemical as well as DNA-based staining methods.

Combined effects of estrogen deficiency and cadmium exposure on calcified hard tissues: Animal model relating to itai-itai disease in postmenopausal women

Using ovariectomized rats as a model of postmenopausal women, we studied the effects of estrogen (Es) deficiency and in combination with cadmium (Cd) exposure on the calcified hard tissues related to the development of itai-itai disease. Es deficiency suppressed the synthesis of carbonic anhydrase required for the crystal nucleation process, causing the crystal structure defects in the tooth enamel. Regarding the combined effects of Es deficiency and Cd exposure on the bone, in which rats were given drinking water containing Cd ions, soft X-ray radiography revealed a development of labyrinthine pattern in the calvaria, and micro-computed tomography demonstrated the declining trabecular architecture of the tibia, suggesting Cd–induced osteoporotic change. Further, electron microscopy showed the increase of amorphous minerals in the calvaria. In conclusion, the combined effects of Es deficiency and Cd exposure can be responsible for accelerating the declining bone strength together with the crystal structure defects resulting in the preferential occurrence of itai-itai disease in postmenopausal women.

Surgical Resection of a Huge Cemento-Ossifying Fibroma in Skull Base by Intraoral Approach

Cemento-ossifying fibroma, also known as ossifying fibroma, usually occurs in the mandible and less commonly in the maxilla. The huge example in the skull base is even rare. We present a case of a huge cemento-ossifying fibroma arising below the skull base of a 30-year-old woman patient. Radiologic investigations showed a giant, lobulated, heterogeneous calcified hard tissue mass, which is well circumscribed and is a mixture of radiolucent and radiopaque, situated at the rear of the right maxilla to the middle skull base. The tumor expands into the right maxillary sinus and the orbital cavity, fusing with the right maxilla at the maxillary tuberosity and blocking the bilateral choanas, which caused marked proptosis and blurred vision. The tumor was resected successfully by intraoral approach, and pathologic examination confirmed the lesion to be a cemento-ossifying fibroma. This case demonstrates that cemento-ossifying fibroma in the maxilla, not like in the mandible, may appear more aggressive because the extensive growth is unimpeded by anatomic obstacles and that the intraoral approach can be used to excise the tumor in the skull base.

Pineal concretions in turkey (Meleagris gallopavo) as a result of collagen-mediated calcification.

The intra-pineal calcification is a well-known phenomenon in mammals, however it is almost completely unknown in birds. The aim of the present work was to analyze morphology and genesis of the pineal concretions in the turkey. The studies were performed on the pineals collected from one-year-old turkeys (Meleagris gallopavo). In addition to standard morphological methods, the alizarin red S and potassium pyroantimonate methods were employed for localization of calcium at the light and electron microscopy level. In light microscopy, calcified concretions with diameters from 300 microm to 2 mm and quantities from 3 to 6 per gland were observed in all the examined pineals. They were stained red with alizarin S and showed the presence of collagen in Mallory's staining. Two types of cells were noted inside the concretion: polygonal and elongated ones. Using electron microscopy, three parts were distinguished within the calcification area. The peripheral part contained densely packed collagen fibrils, some elongated cells and numerous pyroantimonate precipitates demonstrating the presence of calcium ions. In the intermediate part, the fibrils were covered by almost continuous sheets of pyroantimonate precipitates and fused side by side. The central part showed an appearance of calcified hard tissue and contained some polygonal (osteocyte-like) cells. The obtained data demonstrated that the formation of the pineal concretions in the turkey is associated with the mineralization of collagen. This process is completely different from the mechanisms responsible for the formation of the concretions in the mammalian pineal.

Ultrastructure of Apatite Crystals Formed During Vascular Calcification in Humans

Using a transmission electron microscope, we compared the ultrastructure of apatite crystals of normal calcified hard tissue to that of apatite crystals created under pathological conditions. We also sought to verify whether the process of calcification resembles that of osteogenesis after the phenotypic change of soft tissue cells into hard tissue-forming cells. Electron micrographs clearly revealed that cellular debris, plausibly originating from degenerated immune cells like macrophages as well as perivascular cells in intima of femoral artery lesions, seemed to serve in the formation of an organic envelope. This indicates that crystal formation is accompanied by the death of cells. Judging from the lattice image, we also found that the calcified deposits consisted of 2 distinct types of apatite crystals. One type has central dark lines, while the other does not have lines. In addition, the crystals were of various sizes and some showed lattice defects. From these findings, we concluded that ectopic calcification occurred in a manner different from the calcification of normal hard tissues, suggesting that cellular phenotypic change may not happen in vivo.

Differentiation and regenerative capacities of human odontoma-derived mesenchymal cells

Regenerating human tooth ex vivo and biological repair of dental caries are hampered by non-viable odontogenic stem cells that can regenerate different tooth components. Odontoma is a developmental dental anomaly that may contain putative post-natal stem cells with the ability to differentiate and regenerate in vivo new dental structures that may include enamel, dentin, cementum and pulp tissues. We evaluated odontoma tissues from 14 patients and further isolated and characterized human odontoma-derived mesenchymal cells (HODCs) with neural stem cell and hard tissue regenerative properties from a group of complex odontoma tissues from 1 of 14 patients. Complex odontoma was more common (9 of 14) than compound type and females (9 of 14) were more affected than males in our set of patients. HODCs were highly proliferative like dental pulp stem cells (DPSCs) but demonstrated stronger neural immunophenotype than both DPSCs and mandible bone marrow stromal cells (BMSCs) by expressing higher levels of nestin, Sox 2 and βIII-tubulin. When transplanted with hydroxyapatite/tricalcium phosphate into immunocompromised mice, HODCs differentiated and regenerated calcified hard tissues in vivo that were morphologically and quantitatively comparable to those generated by DPSCs and BMSCs. When transplanted with polycaprolactone (biodegradable carrier), HODCs differentiated to form new predentin on the surface of a dentin platform. Newly formed predentin contained numerous distinct dentinal tubules and an apparent dentin–pulp arrangement. HODCs represent unique odontogenic progenitors that readily commit to formation of dental hard tissues.

Carbonic Anhydrase in Calcified Endoskeleton: Novel Activity in Biocalcification in Alcyonarian

Carbonic anhydrase (CA) is a key enzyme in the chemical reaction of living organisms and has been found to be associated with calcification in a number of invertebrates including calcareous sponges, but until now no direct evidence has been advanced to show CA activity in alcyonarian corals. However, it is essential to understand the role of CA in the process of biocalcification in alcyonarian. Here we describe the novel activity of CA and its relationship to the formation of calcified hard tissues in alcyonarian coral, Lobophytum crassum. We find that two CA proteins, which were partially purified by electro-elution treatment, can control the morphology of CaCO(3) crystals and one of them is potentially involved in the process of biocalcification. Previously, we isolated CA from the total extract of alcyonarian, and further, we report here a single protein, which has both calcium-binding and CA activities and is responsible for CaCO(3) nucleation and crystal growth. This matrix protein inhibited the precipitation of CaCO(3) from a saturated solution containing CaCl(2) and NaHCO(3), indicating that it can act as a negative regulator for calcification in the sclerites of alcyonarians. The effect of an inhibitor on the enzyme activity was also examined. These findings strongly support the idea that carbonic anhydrase domain in alcyonarian is involved in the calcification process. Our observations strongly suggest that the matrix protein in alcyonarian coral is not only a structural protein but also a catalyst.

Expression and localization of MGP in rat tooth cementum

Tooth cementum, a calcified hard tissue covering the root surfaces, is an important component connecting the teeth to the collagenous fibres of the periodontal ligament. Although the overall composition of cementum may closely resemble that of bone, each part has not been fully characterized. Here, the localization of the matrix γ-carboxyglutamic acid (Gla) protein (MGP), one of the major Gla-containing proteins in the body, in cementum was investigated using immunohistochemistry and in situ hybridization. (1) Strong MGP antigenicity was observed in the acellular cementum, but was only moderate in the cellular cementum; (2) polygonal periodontal ligament cells facing the acellular cementum and the uncalcified cellular cementum expressed MGP mRNA, indicating that these cells produced MGP and deposited it on the cementum; (3) MGP accumulated at the junction between the uncalcified and calcified cellular cementum; and (4) the distribution pattern of MGP antigenicity resembled that of osteopontin. As one function of MGP could be as a negative regulator for mineral apposition, the expression of MGP in the cells adjacent to the cementum may be important to prevent hyperapposition of minerals.

Purification and structural determination of a phosphorylated peptide with anti-calcification and chitin-binding activities in the exoskeleton of the crayfish, Procambarus clarkii.

Organic matrices in calcified hard tissues have been considered to control calcification. A matrix peptide, designated CAP-1, was extracted and purified by anion-exchange and reverse-phase high performance liquid chromatographies from the exoskeleton of the crayfish, Procambarus clarkii. The amino acid sequence of CAP-1 was determined by mass spectral and sequence analyses of the intact peptide and its enzymatically digested peptides. CAP-1 consisted of 78 amino acid residues, including a phosphoserine residue, and was rich in acidic amino acid residues. CAP-1 had a Rebers-Riddiford consensus sequence, which is conserved in cuticle proteins from many arthropods. CAP-1 inhibited precipitation of calcium carbonate in an in vitro anticalcification assay dose-dependently, and completely inhibited it at 3 x 10(-7) M. CAP-1 also showed chitin-binding ability, indicating that this molecule was bifunctional and played an important role in formation of the exoskeleton.

Biochemical characterization of purified carbonic anhydrase from the octocoral Leptogorgia virgulata

Carbonic anhydrase (CA) has been isolated and purified from the octocoral Leptogorgia virgulata (Lamarck) in an effort to investigate its role in the mineralization and demineralization of spicules and other calcified hard tissues. Affinity-chromatography using Prontosil-derivatized carboxymethylcellulose (CM) Bio-Gel A provided a one-step purification for 30 kdalton polypeptides with carbonic anhydrase activity. Four distinct polypeptides (designated α, β, γ, and δ) are separated from one another at this molecular weight by two-dimensional gel electrophoresis. The specific activity of the L. virgulata CA is on average 57.5±1.5 U g-1, is inhibitable by 10-6 M acetazolamide, and is unaffected by 5mM dithiotheitol. The amino acid composition of these polypeptides is similar to that of mammal, bird, reptile, fish and arthropod species. Antiserum made against the L. virgulata CA reacts specifically with the 30 kdalton polypeptides in western blots, and crossreacts with human CA I and II. Antiserum against avian CA II crossreacts with the L. virgulata 30 kdalton polypeptides. This is the first report of the characterization of a purified CA from an octocoral, and production of a CA antiserum to a species in the phylum Cnidaria.

Effect of the ArF excimer laser on human enamel.

Human enamel surface was irradiated with ArF excimer laser and examined under light microscopy and scanning electron microscopy (SEM). Enamel surface was irradiated at three different areas with different energy fluences. It is demonstrated that the ArF excimer laser causes ablation of the calcified hard enamel tissue. Ablation curves were measured. There was no significant difference found in the etch depth between the three different areas of enamel surface. The morphology of the irradiated areas seen under the SEM was found to be dependent on energy fluence. It changed with increase in energy fluence from being etched to forming a smooth, fused, glaze-like surface and then at very high energy fluences producing a rough surface. The influence of the laser irradiation was confined to the irradiated area only, with no visible heat damage to the surroundings. These results suggest that excimer laser could be applied in a controlled and defined manner for tooth enamel treatments in dentistry.

Analysis of the acoustic response of vascular tissue irradiated by an ultraviolet laser pulse

Stress waves were generated in sections of human vascular tissue by transmitting laser pulses from a XeCl laser source through a glass fiber. Needle-type polyvinylidenefluoride hydrophones were used to detect the acoustic response of the tissue samples during ablation. The experimental arrangement allowed the discrimination in vitro between calcified hard tissue and normal arterial wall immersed in normal saline solution. Atheromatous vessels exhibited a shorter rise time and a higher peak stress than normal tissue.