Low Crystalline Apatite Research Articles

Small organic molecules containing amorphous calcium phosphate: synthesis, characterization and transformation.

As the primary solid phase, amorphous calcium phosphate (ACP) is a pivotal precursor in cellular biomineralization. The intrinsic interplay between ACP and Howard factor underscores the significance of understanding their association for advancing biomimetic ACP development. While organic compounds play established roles in biomineralization, this study presents the synthesis of ACP with naturally occurring organic compounds (ascorbate, glutamate, and itaconate) ubiquitously found in mitochondria and vital for bone remodeling and healing. The developed ACP with organic compounds was meticulously characterized using XRD, FTIR, and solid-state 13C and 31P NMR. The morphological analysis revealed the characteristic spherical morphology with particle size close to 20nm of all synthesized ACP variants. Notably, the type of organic compound strongly influences true density, specific surface area, particle size, and transformation. The in vitro analysis was performed with MC3T3-E1 cells, indicating the highest cell viability with ACP_ASC (ascorbate), followed by ACP_ITA (itaconate). The lowest cell viability was observed with 10 %w/v of ACP_GLU (glutamate); however, 1 %w/v of ACP_GLU was cytocompatible. Further, the effect of small organic molecules on the transformation of ACP to low crystalline apatite (Ap) was examined in Milli-Q® water, PBS, and α-MEM.

Mn導入骨類似組成リン酸カルシウムセメントの創製

Amorphous calcium phosphate (ACP) is known as a precursor of hydroxyapatite (HAp) which is an inorganic main component of bone. The ACP gradually crystallizes to low crystalline apatite near human body temperature. If we introduce an active ingredient at the time of synthesis of ACP and we can introduce an ingredient effective for bone repair or an ingredient having functionality into the crystal structure of HAp, an active ingredient-releasing material responsive to bone metabolism would obtaine, and the protein might produce. Mn is an essential trace element in the living body and is present in the body as an active metal such as Mn-SOD (Mn-superoxmutase) having an antioxidant activity and enhancing the activity of proteoglycan synthetase during bone formation. In this research, we try to introduce Mn into ACP, prepare cement using the obtained powder. In the synthetic powder, Mn was introduced at the same rate as the preparation. The obtained cement had crystallized and low-crytalline bone-like apatite composition. The porosity changed with the amount of Mn added. The cements exhibited protein adsorption properties regardress of the addition of Mn. It is expected that proteins involved in bone metabolism adsorb to the cement.

Periodontal tissue engineering using an apatite/collagen scaffold obtained by a plasma- and precursor-assisted biomimetic process.

In the treatment of severe periodontal destruction, there is a strong demand for advanced scaffolds that can regenerate periodontal tissues with adequate quality and quantity. Recently, we developed a plasma- and precursor-assisted biomimetic process by which a porous collagen scaffold (CS) could be coated with low-crystalline apatite. The apatite-coated collagen scaffold (Ap-CS) promotes cellular ingrowth within the scaffold compared to CS in rat subcutaneous tissue. In the present study, the osteogenic activity of Ap-CS was characterized by cell culture and rat skull augmentation tests. In addition, the periodontal tissue reconstruction with Ap-CS in a beagle dog was compared to that with CS. The plasma- and precursor-assisted biomimetic process was applied to CS to obtain Ap-CS with a low-crystalline apatite coating. The effects of apatite coating on the scaffold characteristics (i.e., surface morphology, water absorption, Ca release, protein adsorption, and enzymatic degradation resistance) were assessed. Cyto-compatibility and the osteogenic properties of Ap-CS and CS were assessed in vitro using preosteoblastic MC3T3-E1 cells. In addition, we performed in vivo studies to evaluate bone augmentation and periodontal tissue reconstruction with Ap-CS and CS in a rat skull and canine furcation lesion, respectively. As previously reported, the plasma- and precursor-assisted biomimetic process generated a low-crystalline apatite layer with a nanoporous structure that uniformly covered the Ap-CS surface. Ap-CS showed significantly higher water absorption, Ca release, lysozyme adsorption, and collagenase resistance than CS. Cell culture experiments revealed that Ap-CS was superior to CS in promoting the osteoblastic differentiation of MC3T3-E1 cells while suppressing their proliferation. Additionally, Ap-CS significantly promoted (compared to CS) the augmentation of the rat skull bone and showed the potential to regenerate alveolar bone in a dog furcation defect. Ap-CS fabricated by the plasma- and precursor-assisted biomimetic process provided superior promotion of osteogenic differentiation and bone neoformation compared to CS.

Bone Tissue Regeneration by Collagen Scaffolds with Different Calcium Phosphate Coatings: Amorphous Calcium Phosphate and Low-Crystalline Apatite.

Surface-mineralized collagen sponges have attracted much attention as scaffolds for bone tissue engineering. Recently, we developed amorphous calcium phosphate (ACP) and low-crystalline apatite coating processes on collagen sponges. In the present study, we applied these coating processes to granular collagen sponges (referred to as Col) to compare the bone tissue regeneration capabilities of ACP-coated and apatite-coated Col (referred to as Col-ACP and Col-Ap, respectively) using a rat cranial bone defect model. According to micro-CT and histological analyses, Col-Ap enhanced bone tissue regeneration compared to Col, whereas Col-ACP did not. These results not only demonstrated the superior bone tissue regeneration capability of Col-Ap, but also indicated limitations of the in vitro simulated body fluid (SBF) test used in our previous study. Despite the apatite-forming ability of Col-ACP in SBF, it was ineffective in improving bone tissue regeneration in vivo, unlike Col-Ap, most likely due to the quick resorption of the ACP coating in the defect site. The present results clarified the importance of the coating stability in vivo and revealed that the low-crystalline apatite coating was more beneficial than the ACP coating in the fabrication of surface-mineralized collagen sponges for use as bone tissue engineering scaffolds.

Spray-drying-derived amorphous calcium phosphate: a multi-scale characterization

Amorphous calcium orthophosphates (ACP) are bioactive compounds presenting high interest as bone substitute. However, the synthesis of such metastable products requires special attention as they can rapidly evolve into a crystalline phase during the elaboration process. The resulting increased stability generally leads to less bioactive reactive materials. Among the various strategies developed to obtain stable form of ACP, the use of spray drying is an effective and reproducible route. Compared to previous works, this study aims to demonstrate for the first time the feasibility of ACP elaboration by spray drying directly from a single solution of selected precursors. Moreover, structuration of the spray-dried powders was determined at different length scales, demonstrating a hierarchical organization from nanometric clusters to particles aggregates. These complementary analyses highlighted a thorough mechanism of particles formation during processing. The effect of the initial composition of the solution was observed, and it was demonstrated that there is a correlation with the purity of the final product that may be modulated. In addition, ACP powders were found to be highly reactive in aqueous medium and their fast transformation into low crystalline apatite suggests a good suitability for biomedical use.

A neoteric procedure for alveolar ridge preservation using autogenous fresh mineralized tooth graft prepared at chair side

BackgroundAlveolar bone loss can occur as a result of aggressive maneuvering due to iatrogenic trauma during teeth extraction or due to normal post-extraction socket healing. Tooth if used as a whole grafting material then successful osteoconductive remodeling can be expected because low-crystalline apatite is the main minerals in bone tissue. A study was conducted in the department of oral and maxillofacial surgery focused on the potential for osteoconduction, osteoinduction, and osteogenesis by growth factors in the tooth and related histogenesis between the tooth and bone. Radiographic evaluation of alveolar ridge preservation was performed using a 3D imaging technique as well as histologically using Autogenous fresh mineralized tooth graft prepared at the chairside. ResultsA total of 30 patients were included in the study. The data was subjected to statistical analysis and the comparison of quantitative data was done using paired t-test. ConclusionThe outcome of this study suggests that chair side autogenous tooth graft can be used for socket survival, sinus and ridge augmentation as it is less time consuming, easy to prepare, with lower bone resorption speed, osteoinduction, osteoconduction, osteogenetic properties, and excellent primary implant stability.

Low crystalline apatite in bone char produced at low temperature ameliorates phosphorus-deficient soils

Globally, more than 30% of soils are poor in phosphorus (P) and the productivity of these soils is severely restricted without the addition of P fertiliser. With future P supplies becoming limited, it is becoming increasingly important to identify ways of optimising the use of waste materials as P fertilisers. One technology that has been promoted extensively in recent years to improve quality of degraded soils is the application of biochar. In this context, char produced from recycled animal bone is of special interest because of its high P content (∼15%). This study investigated how production temperature affects chemical P forms in bone char and the impact on soil P availability in different P-deficient soils.The major P form in dried bone meal was poorly crystalline hydroxyapatite. As the pyrolysis temperature increased to 1050 °C, the hydroxyapatite structure measured with X-ray absorption near edge structure (XANES) spectroscopy persisted. Furthermore, crystallinity increased at temperatures above 750 °C, as revealed by X-ray powder diffraction (XRD). Plant availability was highest for bone char produced between 300 °C and 500 °C in three acidic soils from three continents, and declined rapidly above 750 °C. This strongly indicated that crystallinity of hydroxyapatite limits plant availability at high pyrolysis temperatures. In a high pH soil, all materials resulted in low P availability. As pyrolysis increased the P availability in comparison with dried bone, it was concluded that bone char produced at temperatures between 300 °C and 500 °C has the potential to improve fertility of P-poor, low pH soils.

Structural, crystallographic and morphological properties of microwave-assisted growth of nano-apatite powders

In this study, we present the changes of structures, lattice parameters, crystallinity and morphologies of nano-apatite powders grown via dropwise mixing wet chemical precipitation with microwave treatment for fast synthesis of apatite powders using appropriate amounts of calcium and phosphorous precursors. FTIR, XRD and SEM/EDX were used to characterize the obtained powdered samples. Nano-sized low crystalline and stoichiometric apatite powders comparable to stoichiometric commercial apatite powders were successfully synthesized. The lattice parameters decreased for microwave-treated apatite powders prepared via dropwise addition of phosphorous precursors. The powders exhibited agglomerated nano-sized spherical grains surface morphology.

Bioactive Co-Cr alloy for biomedical applications prepared by surface modification using self-assembled monolayers and poly-γ-glutamic acid.

Cobalt-chromium (Co-Cr) alloys are used in clinical practice for the hard tissue reconstruction because of their favorable biocompatibility and mechanical properties. However, their applications have been limited because of their poor bioactivity, making them poor at bone-bonding. In this study, the bioactivity of a Co-Cr alloy was evaluated following the immobilization of cross-linked poly-γ-glutamic acid (γ-PGA) onto its surface via the formation of 11-aminoundecylphosphonic acid self-assembled monolayers (SAMs). Results of X-ray photoelectron spectroscopy revealed the presence of a new P2p peak, which confirms SAMs formation. Furthermore, the surface became highly hydrophobic following the immobilization with γ-PGA. Subsequent treatment with CaCl2 at 0.5 M or more and soaking in a simulated body fluid led to the formation of a low crystalline apatite. The present results show that chemical modification can be used to induce the formation of an apatite layer on the surface of a Co-Cr alloy in simulated body fluid.

Evaluation of the effects of treatment factors on the properties of bio-apatite materials

Animal bones have been studied as a resource of low-crystalline apatite for immobilization of toxic heavy metals. Chemical and thermal treatments can be applied for selective removal of bone organic constituents. In this study, the impact of different treatment factors was determined by experimental design methodology, and comparison of the estimated effects was performed in order to optimize the treatment conditions. Fractional factorial design with five variables at two levels was applied for factors screening. The type of chemical reagent (H2O2 or NaOH), its concentration (0.1 or 2 mol/L), reaction temperature (20 or 60 °C), contact time (1 or 3 h) and sample annealing (without or at 400 °C) were considered as process variables. Structural properties of bone sorbents, loss of ignition, point of zero charge, surface functional groups, specific surface area, chemical composition, and Cd2+ sorption efficiency, were selected as system responses. Samples significantly differed in respect to the organic phase content and composition, thus mechanisms of Cd2+ sorption were complex. Statistical analysis has revealed that, between all studied factors, temperature treatment was the most important for sorbents increased specific surface area, porosity and cation sorption. Furthermore, NaOH was more effective than H2O2, while the effects produced by variation of other factors were less pronounced. The highest proportion of bio-apatite with desired low crystallinity and high specific surface area was achieved by synergetic effects of chemical treatment with NaOH and thermal treatment at 400 °C.

Synthesis of Zinc-Containing Amorphous Calcium Phosphate

Zinc is an essential trace element in body and has an important role of bone formation. Osteoporosis occurs by imbalance of osteoclasts and osteoblasts activity. The osteoclasts activity lowers the pH of peripheral body environment. Therapeutic agents release material in response to the osteoclasts activity is expected to be a controlled released material. In this study, Zn-containing amorphous calcium phosphate (ACP) powder was synthesized by wet synthesis. Zn content of obtained powders was higher than that of beginning content. ACP powders could be easy to take Zn at wet synthesis. After soaking in phosphate buffered saline for 24 hours, all of synthetic powders were transformed into low crystalline apatite. On the other hand, after soaking in acetic acid buffer for 24 hours, all these powders dissolved. Zn-containing ACP powders are expected to be a Zn controlled released material.

Hydrothermal calcium modification of 316L stainless steel and its apatite forming ability in simulated body fluid

To understand the feasibility of calcium (Ca) modification of type 316L stainless steel (316L SS) surface using hydrothermal treatment, 316L SS plates were treated hydrothermally in calcium chloride (CaCl(2)) solution. X-ray photoelectron spectroscopic analysis revealed that the surface of 316L SS plate was modified with Ca after hydrothermal treatment at 200°C. And the immobilized Ca increased with CaCl(2) concentration. However no Ca-modification was occurred for 316L SS plates treated at 100°C. When Ca-modified 316L SS plate was immersed in simulated body fluid (SBF) with ion concentrations nearly equal to those of human blood plasma, low crystalline apatite was precipitated on its surface whereas no precipitate was observed on non Ca-modified 316L SS. The results obtained in the present study indicated that hydrothermal treatment at 200°C in CaCl(2) solution is useful for Ca-modification of 316L SS, and Ca-modification plays important role for apatite precipitation in SBF.

Firm anchoring between a calcium phosphate-hybridized tendon and bone for anterior cruciate ligament reconstruction in a goat model

Using an alternative soaking process improved the tendon–bone attachment for a calcium phosphate (CaP)-hybridized tendon graft. We characterized the deposited CaP on and in tendons and analyzed the histology and mechanical properties of the tendon–bone interface in anterior cruciate ligament (ACL) reconstruction in goats. The tendon grafts to be implanted were soaked ten times alternately in a Ca-containing solution and a PO4-containing solution for 30 s each. Needlelike CaP nanocrystals including low-crystalline apatite were deposited on and between collagen fibrils from the surface to a depth of 200 µm inside the tendon. The structure resembles the extracellular matrix of bone. In animal experiments, the CaP-hybridized tendon directly bonded with newly formed bone at 6 weeks (n = 3), while fibrous bonding was observed in the control (n = 3). The ultimate failure load was not statistically different between the CaP (n = 7) and control (n = 7). However, in the failure mode, all the tendon–bone interfaces were intact in the CaP group, while three of seven specimens were pulled out from bone tunnels in the control. The result suggested that the strength of the tendon–bone interface in the CaP group is superior to that in the control group. Clinically, firm tendon–bone anchoring may lead to good results without the knee instability associated with the loosening of the bone–tendon junction in ACL reconstruction.

Effect of Saline Solution on the Development of Compressive Strength in Apatite Based Bone Cement Containing Demineralized Bone Matrix

Compressive strength of apatite type bone cement was analyzed with respect to the concentration of setting agent and types of saline solution used for storage after setting. With the increase of the concentration to 2 M, the density of paste decreased. However, the compressive strength of cement was not necessarily correlated with the density and dependent on the type of saline solution used for storage after setting. One solution led rather porous paste and the strongest specimen from the series was obtained under the maximized amount of apatite within the paste. The other saline solution induced the more dense paste after setting. In this series, the strongest specimen was obtained under the coexistence of low crystalline apatite and dicalcium phosphate dihydrate. The difference between two saline solutions was explained in terms of the solidity of skeleton formed by apatite.

Calcium Phosphate Cement (CPC): A Critical Development Path

The first generation of synthetic bone substitute materials (BSM) was initially investigated in the mid 1970s using hydroxyapatite (HA) as a biomaterial for remodeling of bone defects. The concepts established by CPC pioneers in the early 1980s were used as a platform to initiate a second generation of BSM for commercialization. Since then, advances have been made in composition, performance and manufacturing. A self-setting and injectable calcium phosphate cement (CPC) based on amorphous calcium phosphate (ACP) with calcium to phosphate (Ca/P) atomic ratio less than 1.5, combined with dicalcium phosphate dihydrate (DCPD or brushite, seeded with apatite), is proposed. Amorphization of raw material was observed following high energy mechano-chemical processing. Upon hydration, the cement hardened in less than 3 minutes at 37°C and reached a maximum compressive strength of about 50 MPa. The final product was a low crystalline calcium deficient carbonated apatite similar to the composition and structure of bone mineral. In vivo performance of this cement in mediating bone healing was compared to α-BSM® in a rabbit femoral defect model. Performance characteristics of some commercially available CPC products are compared. The concerns of CPC designers and the needs of product users (surgeons) are discussed.

Preparation of Bioactive Poly(Lactic-Co-Glycolic)Acid and Silica Gel Fibers Mixed Non-Woven Fabric

Poly(lactic-co-glycolic)acid and silica gel fibers mixed non-woven fabric was made by electro-spinning method for the potential application as a bone grafting material. The silica gel, the source material for electro-spinning, was prepared by the hydrolysis of tetraethyl orthosilicate in the presence of calcium salt, water, hydrochloric acid and ethanol. Poly(lactic-co-glycolic)acid solution was prepared by dissolving it in the hexafluoroisopropanol. Then, they were transferred to two separate syringes which were connected to the high voltage supply generating a high electric field between the spinneret and the ground collecting drum. The silica gel containing calcium and poly(lactic-co-glycolic)acid solution were spun together under the electric field of 2 ㎸/㎝. The FE-SEM observations showed that the silica gel and poly(lactic-co-glycolic)acid fibers were mixed together completely and its handling property was much improved compared to that of the non-woven silica gel fabric. After soaking in the SBF for 1 week, low crystalline apatite crystals were also observed to occur on the silica fiber surfaces first and then they were also observed to occur on the poly(lactic-co-glycolic)acid fiber surfaces. From the results, it can be concluded that the poly(lactic-co-glycolic)acid and silica gel fibers mixed non-woven fabric made by electro-spinning method has a bioactivity. It means it has a potential to be used as a bone grafting material because of its apatite-forming ability, high surface area to volume ratio and high porosity.

Formation of an ascorbate–apatite composite layer on titanium

An ascorbate–apatite composite layer was successfully formed on NaOH- and heat-treated titanium by coprecipitating L-ascorbic acid phosphate and low-crystalline apatite in a supersaturated calcium phosphate solution at 37 °C for 48 h. The supersaturated calcium phosphate solutions used have chemical compositions attainable by mixing infusion fluids officially approved for clinical use. The amount of immobilized L-ascorbic acid phosphate ranged from 1.0 to 2.3 µg mm−2, which is most likely to be sufficient for the in vitro osteogenic differentiation of mesenchymal stem cells on titanium. Since ascorbate is important for the collagen synthesis and subsequent osteogenesis of mesenchymal stem cells, titanium coated with the ascorbate–apatite composite layer would be useful as a scaffold in bone tissue engineering and as a bone substitute.

Bone resembling apatite by amorphous-to-crystalline transition driven self-organisation

Calcium apatite is the main inorganic constituent of mammalian hard tissues such as bones and teeth. Its formation in vivo is likely to be preceded by a transient amorphous phase. If so, the amorphous-to-crystalline transition would have some crucial role in the biomineralisation process. To investigate this possibility, a two-step biomimetic experiment was designed. First, a stable amorphous calcium apatite precursor was synthesized in simulated body fluid (SBF) and was then transformed into a low crystalline apatite. X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, vacuum FTIR, inductively coupled plasma-atomic emission spectrometry (ICP-AES), scanning electron microscopy (SEM) and N(2) adsorption measurements were used to characterise both the precursor and the apatite. The latter exhibits numerous bone-like features including lack of OH, nanometer size, low crystallinity, etc. An amorphous-to-crystalline transition driven self-organisation is observed. The amorphous precursor seems to be the essential step for the creation of bone resembling apatite.

Calcium Phosphate Biomaterials Composite Device: In-Vitro and In-Vivo Investigation

Low crystalline apatite (LCA) and calcium phosphate cements (CPC) based on amorphous calcium phosphate and dicalcium phosphate dihydrate (1 to 1 ratio) were combined with bioresorbable PLGA copolymer (0 to 20 wt.%) for preparation of solid-formed devices. A pilot manufacturing based on powder processing techniques using isostatic pressure (44,000 psig) was conducted. Processing parameters such as isostatic pressure, temperature, times and device dimension were varied to achieve appropriate mechanical properties comparable to that of allograft bone dowel used as a gold standard in clinical application. The solid-form devices were characterized for physico-chemical and mechanical performance, as well as subjected to an in-vitro wet environment incubation at body temperature (37°C). Fluid diffusion was investigated to evaluate the fluid absorption (through microporosity) and the compressive strength of wet devices vs. incubation time (up to 30 days) was also studied. The shear strength and compressive strength of pure LCA dowels was respectively 26 and 122 MPa, which corresponds to a process densification of about 30%. The compressive strength was dramatically improved with addition of various amounts of copolymer. The maximum compressive strength of 180 MPa was obtained for dowels containing 10 wt.% copolymer. Calcium phosphate composite also increased the shear strength to about 42 MPa. These mechanical performances were significantly higher than that of allograft bone dowel (MD-II™), reported about 10 MPa. A pilot sheep interbody fusion of lumbar spine (L3/L4 and L4/L5) demonstrates mechanical integrity and intervertebral fusion at 6 months. LCA was found to be the most suitable CaP material because of its biocompatibility, chemical composition, nano-structure and high specific surface area that exhibits in-vivo biointegrity and cell mediated process.

Apatite Forming Ability of a Non-Woven Silica Fabric Containing Calcium

Non-woven silica fabric was made by electro-spinning method for the potential application as a bone grafting material. The silica gel, the source material for electro-spinning, was prepared by the hydrolysis of tetraethyl orthosilicate in the presence of calcium salt, water, hydrochloric acid and ethanol. It was transferred to a syringe, which was connected to the high voltage supply generating a high electric field between the spinneret and the ground collecting drum. The silica fibers containing calcium were spun under the electric field of 2 KV/cm. Their diameters were in the range from about 0.3 μm to 8 μm. It was heat-treated at 300 oC for 3 hours. After soaking in the SBF for 1 week, low crystalline apatite crystals were observed to occur on their surfaces. From the results, it can be concluded that the non-woven silica fabric containing calcium made by electro-spinning method and then heat-treated has a bioactivity. It means it has a potential to be used as a bone grafting material because of its apatite-forming ability, high surface area to volume ratio and high porosity.