Hydroxylapatite Crystals Research Articles

Impact of Vertical Stiffness Gradient on the Maximum Divergence Angle.

During vocal fold vibration, the medial surface of both folds forms a convergent shape during opening and a divergent shape during closing. A greater maximum divergence angle is associated with greater closing forces which will increase the closing speed of the glottis. An increased closing speed results in a greater acoustic intensity and greater vocal efficiency. Indentation testing showed that as the strain increases, the inferior aspect of the folds becomes stiffer than the superior aspect, resulting in the vertical stiffness gradient (VSG). We hypothesize that a reduction of the vertical stiffness gradient will reduce the maximum divergence angle. Four excised canine larynges were tested. Stress-strain curves of the superior and inferior aspects of the fold in the mid membranous plane of the baseline larynges were taken using the indentation method. Calcium hydroxylapatite (CaHA) crystals were then injected into the superior aspect of the fold. The stress-strain tests were repeated. Particle imaging velocimetry (PIV) of the intraglottal velocity fields was performed in three larynges at different subglottal pressures in the mid coronal plane for the baseline and CaHA-injected larynges. CaHA injection reduced the inferior-superior stiffness gradient in all larynges. The maximal divergence angle was markedly reduced. In some cases, there was not a divergent angle. Marked reduction of the vertical stiffness gradient significantly reduces the maximum divergence angle. Clinical implications will be discussed. NA Laryngoscope, 131:E1934-E1940, 2021.

Features of basic indicators of oral fluid in children after hematopoietic cell transplantation (pilot study)

Relevance. Treatment of patients with refractory forms of acute lymphoblastic leukemia causes a number of complications from the organs and tissues of the oral cavity. It can be both early complications – cytotoxic reactions with the development of oral mucositis, and late, leading to a violation of the mineralization of hard tooth tissues, and to underdevelopment of the teeth. Oral fluid plays an important role in maintaining oral homeostasis. One of the most important functions that it performs is maintaining the physiological balance of the processes of re- and demineralization of tooth enamel. Since demineralization processes begin in childhood at higher pH values than in adults, when the hydrogen index decreases below 6.4, demineralization processes begin to prevail over remineralization, and the solubility of hydroxylapatite crystals which enamel consists of increases.The objective was to assess the basic indicators of oral fluid and oral hygiene indices in this group of patients.Methods and materials. During the examination of the oral cavity, the intensity of caries and hygiene indices were determined. For the study, oral fluid was collected, followed by determination of its viscosity and hydrogen index.Results. These patients showed a lower level of oral fluid hydrogen index and a lower level of oral hygiene compared to healthy children. In addition, during the study, it was found that there is a direct relationship between the pH of the oral fluid and the time elapsed since the hematopoietic cell transplantation.Conclusion. The detected changes are factors that predispose to an increase in the intensity of caries, and require early preventive measures.Authors declare no conflict of interest.The authors confirm that they respect the rights of the people participated in the study, including obtaining informed consent when it is necessary, and the rules of treatment of animals when they are used in the study. Author Guidelines contains the detailed information.

Polarization induced contrast X-ray fluorescence at submicrometer resolution reveals nanometer apatite crystal orientations across entire tooth sections

For biomedical research, successful imaging of calcified microstructures often relies on absorption differences between features, or on employing dies with selective affinity to areas of interest. When texture is concerned, e.g. for crystal orientation studies, polarization induced contrast is of particular interest. This requires sufficient interaction of the incoming radiation with the volume of interest in the sample to produce orientation-based contrast. Here we demonstrate polarization induced contrast at the calcium K-edge using submicron sized monochromatic synchrotron X-ray beams. We exploit the orientation dependent subtle absorption differences of hydroxyl-apatite crystals in teeth, with respect to the polarization field of the beam. Interaction occurs with the fully mineralized samples, such that differences in density do not contribute to the contrast. Our results show how polarization induced contrast X-ray fluorescence mapping at specific energies of the calcium K-edge reveals the micrometer and submicrometer crystal arrangements in human tooth tissues. This facilitates combining both high spatial resolution and large fields of view, achieved in relatively short acquisition times in reflection geometry. In enamel we observe the varying crystal orientations of the micron sized prisms exposed on our prepared surface. We easily reproduce crystal orientation maps, typically observed in polished thin sections. We even reveal maps of submicrometer mineralization fronts in spherulites in intertubular dentine. This Ca K-edge polarization sensitive method (XRF-PIC) does not require thin samples for transmission nor extensive sample preparation. It can be used on both fresh, moist samples as well as fossilized samples where the information of interests lies in the crystal orientations and where the crystalline domains extend several micrometers beneath the exposed surface.

Mechanical properties, microstructure and morphological properties of badger teeth

The structure, composition and mechanical properties of badger teeth were characterized. At the same time, the difference between badger teeth and human teeth were analyzed. The micrographs of samples showed that badger teeth contained two layers – that is, enamel and dentin. In contrast to hardness, it indicated that the enamel has a higher hardness than dentin, and both are higher than those of humans. Meanwhile, badger teeth have an obvious hardness difference in enamel and dentin. And the badger enamel has a significant anisotropy, the hardness and the elasticity modulus in the cross-section were higher than those in the axial section. Fourier-transform infrared spectroscopy tests show that both the enamel and dentin consisted of carbonate–hydroxylapatite and hydroxyapatite crystals. And X-ray powder diffraction tests show that both the crystal amounts and size of enamel tended to be higher than those of dentin. The water, organic matrix and biomineral of each species were determined by thermogravimetry. Dentin had higher contents of water, organic matrix and carbonate (CO3) than enamel. In the natural world, the mechanical properties of badger teeth are related to their microstructures and compositions, which potentially provide an inspiration for machinery and bionic designs.

Фазовый и гранулометрический состав, структурно-морфологические и текстурные характеристики композиционного сорбента

В работе представлены результаты исследований фазового и гранулометрического состава, морфологических и текстурных характеристик композиционного сорбента на основе бентонитоподобной глины и гидроксилапатита, синтезированного методом химического осаждения. Методами рентгенофазового анализа, ИК-спектроскопии и сканирующей электронной микроскопии зафиксировано образование кристаллов гидроксилапатита в композиционном сорбенте, которые расположены на поверхности частиц минералов, слагающих бентонитоподобную глину. Методом лазерной дифракции определено, что в композиционном сорбенте преобладают частицы размером от 1.0 до 10 мкм. Их содержание достигает 74.76 масс.%. Методом просвечивающей электронной микроскопии установлено, что кристаллы монтмориллонита имеют вид пленок размером 100 – 300 нм, гидроксилапатита – удлиненную форму длиною от 35 до 65 нм и шириною от 12 до 20 нм, размер кристаллов гидроксилапатита в композиционном сорбенте от 8 до 18 нм и от 5 до 8 нм соответственно. Величина удельной поверхности композиционного сорбента, определенная с помощью анализатора TriStar II 3020, равна 96.81 м2/г и больше на 85.78% и 12.85% по сравнению с бентонитоподобной глиной и гидроксилапатитом соответственно.

Antheraea pernyi silk sericin mediating biomimetic nucleation and growth of hydroxylapatite crystals promoting bone matrix formation.

Bone biomineralization is well-regulated processes mediated by extracellular matrix proteins. The materials that can direct nucleation of hydroxylapatite (HAp) crystals and assembly of well-structured material-minerals complex are the key to mimicking the natural mineralization. This study used sericin from Antheraea pernyi (A.pernyi), non-mulberry silkworm cocoon as template to mediate nucleation of HAp crystals. Here we find out that AS (Antheraea pernyi sericin) can nucleate the formation HAp crystals in simulated body fluid verified by XRD and FTIR observations. The HAp crystals are organized into nano-rods oriented with c-axis preferentially parallel to the long axis of AS due to hydrogen bonds and electrostatic interaction and finally aggregated into HAp globule. The cell culture of human bone marrow-derived mesenchymal stem cells (BMSCs) showed that the HAp crystals mediated by AS not only stimulate cell adhesion and proliferation but also promote 0f osteogenic differentiation, suggesting that the resultant mineralized AS biomaterial has potential in promoting bone formation. Thus our work will provide significant implication on biomineralization of A. pernyi silk sericin as a potential scaffold for tissue engineering.

Bio-Templated Growth of Bone Minerals from Modified Simulated Body Fluid on Nanofibrous Decellularized Natural Tissues.

Small intestine submucosal (SIS) membrane used in this study is a decellularized, naturally occurring nanofibrous scaffold derived from a submucosal layer of porcine small intestine. It is predominantly composed of type I collagen fibers. Here we studied the bio-templated growth of hydroxylapatite (HAP) bone minerals on the SIS membrane from a modified simulated body fluid (1.5 SBF) at the body temperature, namely, under a near-physiological condition, in order to evaluate its bone bioactivity, the capability of the membrane in bonding with bone tissue once implanted in vivo. Minute HAP crystals were successfully nucleated on the SIS membranes from 1.5 SBF at the body temperature. The crystals were preferentially nucleated along the collagen fibers constituting the SIS membranes. HAP was the major crystalline mineral phase formed during the whole period of time and a minor crystalline phase of tricalcium phosphate (TCP) appeared after the membranes were incubated for 96 h. We also found that the mineralization for 8 h most significantly promoted the osteogenic differentiation of rat mesenchymal stem cells (MSCs) by evaluating the formation of osteogenic markers in MSCs including alkaline phosphatase (early stage marker) as well as osteocalcin and osteopontin (late stage markers). Hence, SIS membranes show excellent bone bioactivity and once mineralized, can significantly promote the osteogenic differentiation of MSCs.

Biomineralization of Natural Collagenous Nanofibrous Membranes and Their Potential Use in Bone Tissue Engineering.

Small intestinal submucosa (SIS) membranes as a decellularized tissue are known to be a natural nanofibrous biomaterial mainly made of type I collagen fibers and containing some growth factors (fibroblast growth factor 2 and transforming growth factor β) desired in tissue engineering. Here we show that the SIS membranes can promote the formation of bone mineral hydroxylapatite (HAP) crystals along the collagen fibers constituting the membranes from a HAP-supersaturated solution. The resultant biomineralized HAP-SIS scaffolds were found to promote the attachment, growth and osteogenic differentiation of mesenchymal stem cells (MSCs) in both basal and osteogenic media by the evaluation of osteogenic marker formation. More importantly, the HAP-SIS scaffolds could induce the osteogenic differentiation in the basal media without osteogenic supplements due to the presence of HAP crystals in the scaffolds. Histological characterization of the MSC-seeded scaffolds showed that HAP-SIS scaffolds are biocompatible and promote the formation of new tissue in vitro. The biomineralized SIS membranes mimic some aspects of natural bone in terms of the composition and nanostructures and can find potential use in bone tissue engineering.

Natural bone-like biomimetic surface modification of titanium

An implantable metallic surface consisting of titanium (Ti) was modified with natural bone-mimicking CNT–Gelatin–HA nanohybrids to create a new surface with similar properties to the surrounding bone tissue in terms of the chemical constitution, nanotopography, wettability, and biocompatibility. The biomimetic surface modification was achieved through the covalent immobilization of carbon nanotubes (CNTs) onto the Ti surface, the covalent tethering of gelatin molecules onto the CNT surface, and then the deposition of hydroxyl apatite (HA) crystals onto the gelatin-tethered CNTs in SBF solution. The SEM microscopic images demonstrated that the modified Ti surface continually maintained a fibrous structure of CNTs, but that the CNT fibers were hybridized with gelatin and HA in a multi-core-shell structure of similar constitution to that of the collagen fibers of natural bone. The new surface of the Ti substrates showed significantly higher mechanical properties and favorable wettability and biocompatibility.

Replacement of hydroxylapatite by whewellite: implications for kidney-stone formation

AbstractKidney stones consisting predominantly of whewellite (calcium oxalate monohydrate, COM) are often found attached to hydroxylapatite (HA) plaques that form in the soft tissue of kidneys, cause lesions and become exposed to urine. Although the processes of stone formation are not entirely known, it is an established view that so-called Randall’s plaques serve as substrates for COM crystal nucleation and growth from correspondingly supersaturated urine. However, the results presented here suggest an additional mineral replacement process is involved. In an experimental approach, HA was reacted in 0.25 mM, 0.5 mM and 1.0 mM oxalate solutions with pH ranging from 4.5 to 7.5, simulating normal to harsh conditions encountered in urine. Extremely acidic solutions induce dissolution of HA crystals coupled with re-precipitation of the released Ca2+ ions as COM on the HA surface. When, instead, bone was used as a substrate, being more similar to the pathological plaque (aggregated HA nanocrystals within an organic matrix), the HA-COM mineral-replacement reaction is induced even under much milder fluid conditions, commonly found in urine. Hence, a process of COM partly replacing and encrusting Randall’s plaque may take place in the urinary tract of idiopathic stone formers, representing a potential starting event for nephrolithiasis.

Muscle loading is necessary for the formation of a functional tendon enthesis

Muscle forces are essential for skeletal patterning during development. Eliminating muscle forces, e.g., through paralysis, leads to bone and joint deformities. Botulinum toxin (BtxA)-induced paralysis of mouse rotator cuffs throughout postnatal development closely mimics neonatal brachial plexus palsy, a significant clinical condition in infants. In these mice, the tendon-to-bone attachment (i.e., the tendon enthesis) presents defects in mineral accumulation and fibrocartilage formation, presumably impairing the function of the tissue. The objective of the current study was to investigate the functional consequences of muscle unloading using BtxA on the developing supraspinatus tendon enthesis. We found that the maximum endurable load and stiffness of the supraspinatus tendon attachment decreased after four and eight weeks of post-natal BtxA-muscle unloading relative to controls. Tendon cross-sectional area was not significantly reduced by BtxA-unloading, while, strength, modulus, and toughness were decreased in the BtxA-unloaded group compared to controls, indicating a decrease in tissue quality. Polarized-light microscopy and Raman microprobe analysis were used to determine collagen fiber alignment and mineral characteristics, respectively, in the tendon enthesis that might contribute to the reduced biomechanical performance in BtxA-unloaded shoulders. Collagen fiber alignment was significantly reduced in BtxA-unloaded shoulders. The mineral-to-matrix ratio in mineralized fibrocartilage was not affected by loading. However, the crystallographic atomic order of the hydroxylapatite phase (a measure of crystallinity) was reduced and the amount of carbonate (substituting for phosphate) in the hydroxylapatite crystals was increased. Taken together, these micrometer-scale structural and compositional changes partly explain the observed decreases in the mechanical functionality of the tendon enthesis in the absence of muscle loading.

Silk fibroin derived polypeptide-induced biomineralization of collagen

Silk fibroin (SF) is extensively investigated in osteoregenerative therapy as it combines extraordinary mechanical properties and directs calcium-phosphate formation. However, the role of the peptidic fractions in inducing the protein mineralization has not been previously decoded. In this study, we investigated the mineralization of fibroin-derived polypeptides (FDPs), which were obtained through the chymotryptic separation of the hydrophobic crystalline (Cp) fractions and of the hydrophilic electronegative amorphous (Cs) fractions. When immersed in simulated body fluid (SBF), only Cs fragments demonstrated the formation of carbonated apatite, providing experimental evidence that the mineralization of SF is dictated exclusively by its electronegative amino-acidic sequences. The potential of Cs to conceptually mimic the role of anionic non-collagenous proteins in biomineralization processes was investigated via their incorporation (up to 10% by weight) in bulk osteoid-like dense collagen (DC) gels. Within 6 h in SBF, apatite was formed in DC-Cs hybrid gels, and by day 7, carbonated hydroxylapatite crystals were extensively formed. This accelerated 3-D mineralization resulted in a nine-fold increase in the compressive modulus of the hydrogel. The tailoring of the mineralization and mechanical properties of hydrogels through hybridization with FDPs could potentially have a significant impact on cell delivery and bone regenerative medicine.

Self-Assembly and Mineralization of Genetically Modifiable Biological Nanofibers Driven by β-Structure Formation

Bioinspired mineralization is an innovative approach to the fabrication of bone biomaterials mimicking the natural bone. Bone mineral hydroxylapatite (HAP) is preferentially oriented with c-axis parallel to collagen fibers in natural bone. However, such orientation control is not easy to achieve in artificial bone biomaterials. To overcome the lack of such orientation control, we fabricated a phage-HAP composite by genetically engineering M13 phage, a nontoxic bionanofiber, with two HAP-nucleating peptides derived from one of the noncollagenous proteins, Dentin Matrix Protein-1 (DMP1). The phage is a biological nanofiber that can be mass produced by infecting bacteria and is nontoxic to human beings. The resultant HAP-nucleating phages are able to self-assemble into bundles by forming β-structure between the peptides displayed on their side walls. The β-structure further promotes the oriented nucleation and growth of HAP crystals within the nanofibrous phage bundles with their c-axis preferentially parallel to the bundles. We proposed that the preferred orientation resulted from the stereochemical matching between the negatively charged amino acid residues within the β-structure and the positively charged calcium ions on the (001) plane of HAP crystals. The self-assembly and mineralization driven by the β-structure formation represent a new route for fabricating mineralized fibers that can serve as building blocks in forming bone repair biomaterials and mimic the basic structure of natural bones.

Crystallographic Control of the Hydrothermal Conversion of Calcitic Sea Urchin Spine (Paracentrotus lividus) into Apatite

We analyzed the crystallographic relationships during hydrothermal conversion of a calcitic sea urchin spine into apatite. We identified a pseudomorphic mineral replacement mechanism involving a superficial dissolution of calcite and a subsequent overgrowth of oriented carbonated hydroxylapatite (HA) nanocrystals. Cross-section images of these converted spines show that the dimensions of the HA crystals increase the further they are from the outer surface. This replacement process is favored by an increase in porosity, which enables both fluid and mass to be transported by diffusion, thereby allowing the replacement reaction to progress toward the interior of the spine. These recrystallization reactions take place on the surface of the calcite single crystal, which acts as a substrate for the epitaxial nucleation of HA crystals. The epitaxial relationship observed between the parent calcite crystal and the newly formed apatite crystals can be defined as (0001) apatite//(011̅8) calcite and [10.0] apatite//[4̅4.1] calcite. The apatite crystals are related by the 3-fold axis arising from the trigonal symmetry of the parent calcite crystal. There is therefore a strong structural control which favors the conversion of calcite into apatite. This process coexists with the formation of apatite crystals which are not structurally related to the calcite crystal and which may precipitate within the porosity of the material. The analysis of crystallographic relationships is a fundamental step toward understanding mineral replacement reactions, which can be used for the synthesis of artificial materials with predefined shapes and microstructural characteristics, a technique that may have interesting technological applications.

Hydroxyl-apatite crystals in the urine of stanniectomized freshwater North American Eels (Anguilla rostrata LeSueur)

A white micro-crystalline precipitate was observed in the urine of freshwater North American eels from 7-16 days after Stanniectomy. Scanning electron microscopy and energy dispersive x-ray microanalysis showed that the amorphous crystals contained calcium and phosphorous but no magnesium or other elements. Subsequent x-ray diffraction analysis showed that the crystals were hydroxyl-apatite. These crystals are formed in urine as a consequence of the increased urinary excretion of calcium that always follows removal of the corpuscles of Stannius.

Role of 20-kDa Amelogenin (P148) Phosphorylation in Calcium Phosphate Formation in Vitro

The potential role of amelogenin phosphorylation in enamel formation is elucidated through in vitro mineralization studies. Studies focused on the native 20-kDa porcine amelogenin proteolytic cleavage product P148 that is prominent in developing enamel. Experimental conditions supported spontaneous calcium phosphate precipitation with the initial formation of amorphous calcium phosphate (ACP). In the absence of protein, ACP was found to undergo relatively rapid transformation to randomly oriented plate-like apatitic crystals. In the presence of non-phosphorylated recombinant full-length amelogenin, rP172, a longer induction period was observed during which relatively small ACP nanoparticles were transiently stabilized. In the presence of rP172, these nanoparticles were found to align to form linear needle-like particles that subsequently transformed and organized into parallel arrays of apatitic needle-like crystals. In sharp contrast to these findings, P148, with a single phosphate group on serine 16, was found to inhibit calcium phosphate precipitation and stabilize ACP formation for more than 1 day. Additional studies using non-phosphorylated recombinant (rP147) and partially dephosphorylated forms of P148 (dephoso-P148) showed that the single phosphate group in P148 was responsible for the profound effect on mineral formation in vitro. The present study has provided, for the first time, evidence suggesting that the native proteolytic cleavage product P148 may have an important functional role in regulating mineralization during enamel formation by preventing unwanted mineral formation within the enamel matrix during the secretory stage of amelogenesis. Results obtained have also provided new insights into the functional role of the highly conserved hydrophilic C terminus found in full-length amelogenin.

Nanostructured 3-D collagen/nanotube biocomposites for future bone regeneration scaffolds

The field of bionanotechnology has been rapidly growing during the last few years and we can now envision a controllable integration between biological and artificial matter, where new biomimetic structures with a wide range of chemical and physical properties will promote the development of a novel generation of medical devices. In this work we describe a collagen/carbon nanotube composite which has the potential to be used as a scaffold for tissue regeneration. Because this biocomposite incorporates the advantageous properties of both collagen and carbon nanotubes, it has most of the characteristics that an ideal biomaterial requires in order to be used as an osteoinductive agent. This biocomposite is bioresorbable and biodegradable and has the desired mechanical rigidity while maintaining a three-dimensional(3-D) nanostructured surface. Tuned stability and swelling were achieved under fluid environments by varying the amount of carbon nanotubes (CNTs) incorporated into the composite. These variations can dictate the degree of interaction between fibroblastic cells and the biomaterials. Proof-of-concept was shown by performing an in vitro induced mineralization of hydroxylapatite crystals under physiological conditions. Furthermore, the ability to attach biofunctional groups to the CNT walls can open a new road for tissue regeneration since the combination of CNTs with specific growth factors or cellular ligands can create an environment capable of signaling and influencing specific cell functions. Our observations suggest that collagen/carbon nanotube biocomposites will have important uses in a wide range of biotechnological areas.

Radiesse-Induced Herpes Zoster

Radiesse is a filler material for deep nasolabial folds that was recently approved by the United States Food and Drug Administration. It is composed of calcium hydroxylapatite crystals measuring 25 mum to 45 mum. Few complications have been reported to date. We present a case of a woman who developed herpetic appearing skin lesions after the injection of Radiesse in the glabella. She noted tenderness and tingling along with redness and bumps. The vesicles were isolated to the right ophthalmic branch of the trigeminal nerve distribution and the skin was erythematous with associated pustules. After disease progression while on systemic antibiotics, she improved markedly when started on antivirals. Three days later the tingling resolved, the erythema was markedly improved, and the vesicles started to resolve. She had returned to baseline after a month. Physicians should be suspicious for herpetic infection or reactivation with facial injections of Radiesse or other fillers and should initiate immediate treatment with oral antivirals upon identifying the above signs and symptoms.

Ultrastructure of detritus synovitis ("hydroxylapatite synovitis?") (author's transl)

Detritus synovitis is a sequel of cartilage and osseous destruction in diarthrosis. Cartilage and bone fragments are enclosed by granulation tissue. Remnants of the destroyed bone and calcified cartilage appear as defined accumulations of hydroxylapatite crystals in the granulation tissue. These hydroxylapatite tissues resemble those considered by other authors as being responsible for a "third crystal synovitis, namely, hydroxylapatite synovitis". However, the findings communicated here allow us to conclude that these crystals are the sequel of a destruction of diarthrosis, so that it is not justified to consider hydroxylapatite synovitis as a separate disease entity.

Oriented Nucleation of Hydroxylapatite Crystals on Spider Dragline Silks

Spider dragline silk as a protein fiber can be pictured as the oriented organization of protein nanocrystals along the long axis with their spacing filled by amorphous protein domains. We used the surface of the spider dragline silk as a biological template to nucleate bone mineral hydroxylapatite (HAP) site-specifically from a HAP-supersaturated solution. HAP crystals were found to be nucleated on the surface of silks with their c axis preferentially oriented at an average angle of 72.9 degrees with respect to the long axis of the silks. The preferred orientation is nearly identical among the different mineralized silks that we studied. Other materials such as Au and CdS could be nucleated on the silks but did not show any preferred orientation. We believe that the oriented nucleation of HAP is directly related to the structures of silks and HAP. The mineralized silks will combine the good mechanical properties of the spider silks and the biocompatibility of HAP and may be assembled into ideal biomaterials as bone implants.