Composite material based on bioactive glass and amorphous calcium phosphate and its manufacturing method

Abstract

The present invention relates to an amorphous calcium phosphate / bioactive glass composite material (ACP / BG) that combines the short-term ion-releasing ability of ACP with the long-term ion-releasing ability of BG, thereby (i) efficiently remineralizing damaged enamel and (ii) inducing the formation of a new HA layer on the surface of enamel and dentin. The ACP / BG composite material is obtained by embedding BG particles in a matrix of ACP nanoparticles to maximize homogenization between these two components and produce a composite material with high uniformity from the macroscale to the nanoscale. This is achieved by a method including precipitating ACP onto BG particles. The BG particles can be nanoparticles, microparticles, or macroparticles. The particles can also be porous or mesoporous. The present invention also relates to the use of the composite material of the present invention for preventing demineralization and promoting remineralization of teeth. In other words, the composite material disclosed herein is 2+ ions and PO4 3- By releasing ions, it has the ability to restore the structure and maintain the mechanical properties of dental hard tissues, and it can be used as a remineralization agent, mimicking the spontaneous remineralization process induced by saliva. The composite material can also be used for bone regeneration.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a composite material comprising or consisting of amorphous calcium phosphate and bioactive glass, and to its use as a remineralizing agent in dentistry and for bone regeneration.

[0002] According to the 2017 World Disease Survey, dental caries is the most prevalent non-communicable disease affecting both developed and developing countries. The disease is caused by the prevalence of acid-producing bacteria within a microbial film that permanently colonizes oral surfaces. These cariogenic bacteria produce organic acids, which result in a decrease in the pH of the microenvironment of the tooth surface (enamel and dentin), damaging the mineral components hydroxyapatite (HA, Ca). 10 This causes the dissolution of HA (PO4)6(OH)2). The dissolution of HA is called the "demineralization process" and leads to the formation of cavities. Demineralization is a reversible process if the damaged tissue is exposed to an oral environment that promotes the opposite process, "remineralization." Demineralized enamel can be repaired by saliva, which then absorbs Ca 2+ ions and PO4 3- Natural remineralization occurs through epitaxial growth of residual HA crystals, which act as nucleation sites to provide a supersaturated environment rich in ions. However, saliva alone rarely achieves remineralization of enamel. Therefore, to effectively inhibit demineralization and promote remineralization, a Ca-rich solution is required. 2+ ions and PO4 3- It is necessary to increase the ionic supersaturation using an external source of ions. 2+ ions and PO4 3- The purpose of remineralizing agents is to restore the structure and maintain the mechanical properties of dental hard tissues by releasing ions. [1] Modern dentistry is increasingly focused on early caries prevention rather than invasive restorative treatments, which has led to increased interest in remineralizing agents. In fact, the best way to prevent caries is to inhibit the formation and progression of caries by promoting remineralization of tooth surfaces.

[0003] Various forms of synthetic calcium phosphates have been proposed for this purpose [2]. These materials mimic the composition and structure of the mineral phase of teeth and therefore 2+ Ions and PO4 3- Ideal for releasing ions. The synthetic calcium phosphates most commonly used for tooth remineralization are HA, fluorhydroxyapatite (FHA), and amorphous calcium phosphate (ACP). These calcium phosphate agents can be added to restorative materials or applied directly to the tooth surface.

[0004] Among these calcium phosphate agents, ACP is particularly attractive in dentistry because it can release more ions compared to crystalline calcium phosphate phases. Several ACP products are actually marketed for tooth remineralization in formulations applied directly to the tooth surface (e.g., dental mousses), and there is a sufficient level of literature evidence (reviews) for this use. ACP is an unstable substance that rapidly transforms into a more thermodynamically stable crystalline phase (e.g., HA) when reacting with atmospheric moisture in solution or in the dry state. This instability makes ACP difficult to use and handle, and several additives have been investigated to stabilize ACP. In this regard, WO2020 / 002517 and W2016 / 012452 describe fluoride-doped amorphous calcium phosphate (F-ACP) stabilized with citrate ions, which in aqueous solution exhibits a high ionic strength. 2+ ions, PO4 3- ions, and F - They release large amounts of ions, crystallize into HA upon contact with enamel and dentin, inhibit the formation of cariogenic microbial films, and exhibit potent remineralization activity. Therefore, known F-ACPs have proven to be excellent materials for tooth remineralization. The main limitation of all ACP materials (F-ACP or commercial ACP products) is that their large-scale release lasts only for a short time. Therefore, multiple applications are required for complete remineralization.

[0005] Another major class of materials currently used as remineralization agents is bioactive glass (BG). BG is well-known as a material for hard tissue regeneration due to its bioactivity (HA formation ability), biocompatibility, osteoconductivity, and osteoinductivity, as well as its controlled release of bioactive ions into the surrounding environment. Bioactive glass nanoparticles (BGN) have also attracted increasing attention due to their unique properties, such as high bioactivity and higher loading and release of ions, drugs, and other biomolecules, owing to their small size and large specific surface area. An advantage of BG and BGN materials is that they can be manufactured with a wide range of ion substitutions, allowing for the release of many bioactive ions over time. As a result of their controlled ion release ability, BG has been successfully used as a remineralization agent. However, a significant drawback hindering this application is that BG has a low Ca content. 2+ ions and PO4 3- Because it cannot release large amounts of ions and they are quickly diluted by the action of saliva before they can adhere to the tooth surface, it is unable to fully perform the remineralization function required to form HA.

[0006] Therefore, considering the latest technology in remineralization agents, the remineralization ions (Ca) 2+ , PO4 3‐ ) and bioactive ions (e.g., F - , Si 4+ , Sr 2+ , Zn 2+ , Mg 2+ There is an unmet need for materials that release HA and simultaneously induce the formation of new HA crystals on the worn enamel and dentin. Summary of the Invention

[0007] The present invention relates to an amorphous calcium phosphate / bioactive glass composite material (ACP / BG). The composite material combines the short-term ion-releasing ability of ACP with the long-term ion-releasing ability of BG, and therefore (i) efficiently remineralizes damaged enamel and (ii) induces the formation of a new HA layer on the surface of enamel and dentin. Specifically, the ACP component has two functions: (i) it provides ions (Ca) for the initial HA formation;2+ , PO4 3- , F - and Sr 2+ , Mg 2+ , Zn 2+ ), and (ii) attach to the surfaces of enamel and dentin to act as nucleation sites for HA growth. Meanwhile, the ion-doped BG components release remineralization ions and bioactive ions (Ca 2+ , PO4 3- , F - , Si 4+ , Sr 2+ , Zn 2+ , Mg 2+ This results in a continuous release of ions, which leads to continuous remineralization.

[0008] The ACP / BG composite material is obtained by embedding BG particles in a matrix of ACP nanoparticles to maximize homogenization between these two components and produce a composite material with high uniformity from the macroscale to the nanoscale. This is achieved by a method including precipitating ACP onto BG particles. The BG particles can be nanoparticles, microparticles, or macroparticles. The particles can also be porous or mesoporous.

[0009] The present invention also relates to the use of the composite material of the present invention for preventing demineralization and promoting remineralization of teeth. In other words, the composite material disclosed herein is 2+ ions and PO4 3- It is used as a remineralizing agent because it has the ability to restore the structure and maintain the mechanical properties of dental hard tissues by releasing ions, and it mimics the spontaneous remineralization process induced by saliva.

[0010] The composite material can also be used for bone regeneration. [Brief explanation of the drawings]

[0011] Figure 1(A-H): SEM micrographs of (A) ACP / BG 80 / 20 composite material, (B) ACP / BG 60 / 40 composite material, (C) ACP / BG 40 / 60 composite material, (D) ACP and BG physical mixture, (E) ACP / MBGN 80 / 20 composite material, (F) ACP / MBGN 60 / 40 composite material, (G) ACP / MBGN 40 / 60 composite material, and (H) ACP and MBGN physical mixture. Light gray arrows indicate BG or MBGN particles, and black arrows indicate ACP particles.

[0012] Figure 2: PXRD patterns of (A) ACP / BG composite material and (B) ACP / MBGN composite material. FT-IR spectra of (C) ACP / BG composite material and (D) ACP / MBGN composite material.

[0013] Figure 3 (A-G): Cumulative ion release of (A) Ca, (C) P, (E) Si, and (G) F from ACP / BG composite materials and physical mixtures, and cumulative ion release of (B) Ca, (D) P, (F) Si, and (H) F from ACP / MBGN composite materials and physical mixtures.

[0014] Figure 4: SEM micrographs of (A) ACP / MBGN 80 / 20, (B) ACP / MBGN 60 / 20, (C) ACP / MBGN 40 / 60, (D) pure MBGN, and (E) pure ACP after 7 days of incubation in SBF.

[0015] The composite material of the present invention comprises or consists of particles of bioactive glass and particles of amorphous calcium phosphate in a homogeneous and intimate mixture, which is obtained by precipitating ACP onto the BG particles.

[0016] The BG particles are selected from macroparticles, microparticles, or nanoparticles, each of which may be microporous or mesoporous. BG nanoparticles are defined as BGNs, and mesoporous BG nanoparticles are defined as MBGNs.

[0017] The compositions of macro and micro BG particles typically contain SiO2, CaO, Na2O, and P2O5 in different proportions, and other components such as ZnO, CuO, and SrO can also be incorporated into the BG composition.

[0018] BGN and MBGN compositions typically contain different ratios of SiO2 and CaO. For example, BGN is composed of 90% SiO2 and 10% CaO, while MBGN is composed of 10% CaO and 90% SiO2. Other components, such as ZnO, CuO, and SrO, can also be incorporated into BG nanoparticles and MBGN.

[0019] Preferably, the ACP is in the form of nanoparticles.

[0020] In one embodiment of the present invention, the ACP is doped with one or more of the following ions: F, Zn, Mg, Sr, Na, K, Fe, Cu, Cl, and Ag.

[0021] The composite material contains ACP in the range of 1 wt % to 99 wt % and BG in the range of 99 wt % to 1 wt %.

[0022] In another embodiment, the composite material comprises ACP in the range of 20 wt% to 80 wt%, or 20 wt% to 60 wt%, or 20 wt% to 40 wt%, and BG in the range of 80 wt% to 20 wt%, or 60 wt% to 20 wt%, or 40 wt% to 20 wt%.

[0023] Both components are intimately and uniformly entangled, resulting in the composite material exhibiting properties superior to those of a simple mixture of both components, ACP and BG.

[0024] The composite material avoids the inherent shortcomings of its constituent components, such as poor short-term ion release, limited apatite formation, no fluoride release in BG, and no sustained release and poor stability in ACP.

[0025] Said composite material is obtained by a method comprising the following steps: a) mixing an aqueous solution (Solution A) containing calcium salt and / or calcium hydroxide, BG particles, and sodium citrate or potassium citrate and / or citric acid with a solution (Solution B) containing phosphate and / or phosphoric acid, and carbonate and / or carbonic acid; b) stirring the mixture for at least 3 seconds to form a precipitate; and c) collecting and drying the precipitate.

[0026] The calcium salt is selected from calcium chloride, calcium nitrate, calcium acetate, calcium lactate, calcium oxalate, calcium citrate, and calcium sulfate.

[0027] The sodium or potassium citrate salt is selected from trisodium citrate, disodium citrate, monosodium citrate, tripotassium citrate, dipotassium citrate, and monopotassium citrate.

[0028] The phosphate salt is selected from sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, or potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, or ammonium phosphate, ammonium hydrogen phosphate, and ammonium dihydrogen phosphate.

[0029] The carbonate is selected from sodium carbonate, sodium bicarbonate, sodium dihydrogen carbonate, or potassium carbonate, potassium bicarbonate, potassium dihydrogen carbonate, or ammonium carbonate, ammonium bicarbonate, and ammonium dihydrogen carbonate.

[0030] In step b), the mixture is stirred for 3 seconds to 1 hour, preferably 10 seconds to 60 seconds.

[0031] The precipitate is collected, for example, by centrifugation, filtration, vacuum filtration, or decantation, and then dried, for example, by freeze drying, heating, or spray drying.

[0032] BG particles (BG macroparticles or BG microparticles, BGN, or MBGN) are prepared by methods known in the art or are commercially available.

[0033] Prior to step a), solution A can be optionally sonicated to disperse the BG particles, and the pH of solution B can optionally be made basic, greater than 9.

[0034] The amount of BG particles used can be varied to give a final ACP:BG weight ratio of, for example, 80:20, 60:40, or 40:60.

[0035] Solution A can optionally contain zinc, strontium, iron, silver, copper, sodium, potassium, and / or magnesium ions, which are doping agents for the ACP.

[0036] Solution B can optionally contain fluorine and / or chlorine ions, which are doping agents for the ACP.

[0037] The mixing step a) is preferably carried out at a temperature of 1 to 30°C, or 4 to 25°C, or 20 to 25°C.

[0038] The composite material obtained by the method of the present invention has a structure in which BG particles are surrounded and immersed in a matrix of ACP nanoparticles.

[0039] There is no separation between ACP and BG, and the mixture is homogeneously and intimately mixed to form a deeply entangled composite material.

[0040] The relative abundance of BG microparticles in the composite material decreases with increasing ACP:BG ratio, confirming the ability to control the composition of the composite material.

[0041] Comparing the physical mixture of ACP and BG with the composite material of the present invention shows that the physical mixture is not homogeneous but exhibits microgranules of pure BGN / MBGN or pure ACP.

[0042] The composite material of the present invention, when doped with one or more of Ca, P, Si, and optionally F, Zn, and / or Mg ions, exhibited good release of these ions. The release was stronger for at least the first two hours and continued for a second period of up to five hours. Thereafter, the release gradually slowed down, resulting in a gradual release for up to 24 hours.

[0043] Comparison of the release curves shows that pure ACP only shows a massive release of Ca, P, and F within 2 h, while pure BG shows less intense release of Ca, P, and Si, but they are distributed over 24 h.

[0044] For the ACP / BG composite materials, the release was intermediate between those of pure ACP and pure BG and depended on the ACP:BG ratio. Specifically, composite materials with a high ACP content exhibited stronger Ca, P, and F release and weaker Si release. On the other hand, composite materials with a high BG content exhibited the opposite behavior. This indicates that ion release can be tuned by adjusting the ACP:BG ratio. Most importantly, the ACP / BG composite materials exhibited stronger Ca, P, and F release and weaker Si release compared to a physical mixture of ACP and BG at the same weight ratio. This clearly demonstrates that the preparation of this composite material can achieve superior ion release properties that cannot be achieved by simply mixing the two basic components.

[0045] In the case of the ACP / MBGN composite material, the observed behavior is the same, but the differences between the composite material, pure MBGN, and the physical mixture of ACP and MBGN are smaller.

[0046] Tests conducted by applicants have demonstrated that the composite materials of the present invention can be used to prevent tooth demineralization and promote remineralization, which are useful in the prevention and treatment of caries and tooth decay.

[0047] The composite materials of the present invention can also be used to reduce dentin hypersensitivity by occluding dentin tubules.

[0048] Another application of the composite material of the present invention is bone regeneration.

[0049] This composite material can also be used for bone regeneration. Due to its excellent remineralization and ion release capabilities, this composite material can be used as a filler for bone defects, promoting HA formation and osteogenesis and angiogenesis. This composite material can also be used to coat orthopedic implants for bone repair and regeneration or to fabricate scaffolds for bone tissue engineering. Ca from ACP 2+ ions and PO4 3- The rapid release of ions promotes osseointegration, while the sustained release of bioactive ions from BG ensures a favorable biological response for bone regeneration.

[0050] The present invention also relates to a method for preventing tooth demineralization and / or promoting tooth remineralization, comprising applying the composite material to dental hard tissue.

[0051] Materials and Methods Materials and Precursors Reagents required for the preparation of the composite material: Calcium chloride dihydrate (CaCl2·2H2O, purity 99.0% or more), hydrochloric acid (HCl, purity 37.0% or more), sodium citrate trihydrate (Na3(C6H5O7) ·2H2O, purity 99.0% or more, hereafter referred to as sodium citrate), disodium phosphate dihydrate (Na2HPO4·2H2O, purity 99.0% or more), sodium carbonate monohydrate (Na2CO3·2H2O, purity 99.0% or more), sodium fluoride (NaF, purity 99.0% or more), MBGN, BGN, BG.

[0052] MBGN was synthesized and characterized using a microemulsion-based sol-gel method according to the method reported by Zheng et al. [3]. In particular, in a typical procedure, 0.56 g of cetrimonium bromide (CTAB, > 97%, Sigma-Aldrich) was dissolved in 26 mL of deionized water with stirring. After CTAB was completely dissolved, 8 mL of ethyl acetate ( > After stirring for 30 min, 5.6 mL of ammonia solution (1 M, VWR) was added. After stirring for another 15 min, 2.88 mL of tetraethyl orthosilicate (TEOS, > 99.0%, Sigma-Aldrich) and 1.83 g of calcium nitrate tetrahydrate ( > The precipitate was then dried overnight at 60°C and then calcined at 700°C for 4 hours at a heating rate of 2°C / min.

[0053] BGN nanoparticles were synthesized using a modified Stober method (Zheng K et al., The timing of calcium nitrate addition affects the morphology, dispersion, and composition of bioactive glass nanoparticles. [J]. RSC Advances, 2016, 6(97): 95101-95111). In a typical synthesis procedure, solution A, consisting of 2.25 mL of tetraethyl orthosilicate (TEOS, 98%, Sigma-Aldrich) and 25 mL of ethanol (96% VWR), was mixed with solution B, which was prepared by mixing 4.5 mL of ammonium hydroxide solution (28.0–30.0%, Sigma-Aldrich), 8.12 mL of ethanol, and 12.38 mL of deionized water. After allowing the reaction to stand for 30 minutes, 1.45 g of calcium nitrate tetrahydrate was added. This mixture was allowed to react for an additional 90 minutes before being collected by centrifugation at 7197 rcf for 25 minutes. The resulting particles were dispersed and washed twice with deionized water and once with ethanol. The collected particles were dried at 60 °C overnight and then calcined at 700 °C for 2 h at a heating rate of 2 °C / min.

[0054] Commercially available "45S5 type" BG microparticles were obtained from Schott Glass. Product name: Schott Vitryxx bioactive glass powder, size: SM4.0, material: MD01.

[0055] Preparation of ACP / BG complex ACP / BG complexes were prepared by mixing equal volumes of two aqueous solutions at room temperature: (A) 100 mM CaCl2 + 100 mM sodium citrate + X mg / mL BG (either BG, BGN, or MBGN) and (B) 120 mM Na2HPO4 + 200 mM Na2CO3 + 50 mM NaF. X was optimized to achieve a final ACP:BG weight ratio of 80:20, 60:40, or 40:60 (where X = 2 mg / mL for 80 / 20 ACP / BG; X = 6 mg / mL for 60 / 40 ACP / BG; and X = 18 mg / mL for 40 / 60 ACP / BG). Before mixing, solution (A) was sonicated with a Vibracell VCX 500 tip sonicator (SONICS, Newtown, Connecticut, USA) for 3 minutes at 20% amplitude and 5-second pulses under ice cooling to disperse BG. Meanwhile, solution (B) was adjusted to pH 9.5 with 37 wt% hydrochloric acid. After mixing, the precipitate was stirred at room temperature for at least 30 seconds, and then the particles were collected by centrifugation (7000 RPM, 4°C for 5 minutes) and washed repeatedly with ultrapure water. Finally, the material was freeze-dried for 24 hours.

[0056] Preparation of control composite material A 60:40 wt% physical mixture of ACP and BG was prepared by manually mixing 240 mg of ACP powder with 160 mg of BG powder (either BG, BGN, or MBGN) until completely homogenous.

[0057] Chemical, morphological, and structural characterization Powder X-ray diffraction (PXRD): PXRD patterns of the samples were recorded on a D8 Advanced Diffractometer (Bruker, Karlsruhe, Germany) using Cu Kα radiation generated at 40 kV and 40 mA. PXRD patterns were collected for 0.5 s in the range of 10–60°2θ with a step size of 0.02°.

[0058] Fourier transform infrared spectroscopy (FT-IR): FT-IR spectra were collected on a Nicolet 5700 spectrometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) equipped with an ATR iD7 accessory. Spectra were collected in ATR mode, 4000–400 cm. -1 32 scans in the range of 1000 and a resolution of 2 cm -1 Collected in.

[0059] Specific surface area analysis (SSA BET ): The specific surface area of ​​the samples was measured by N2 gas adsorption and Brunauer-Emmett-Teller (BET) methods using a Sorpty 1750 apparatus (Carlo Erba, Milan, Italy).

[0060] Thermal characterization: Thermogravimetric analysis (TGA) of the samples was performed using a STA 449F3 Jupiter instrument (Netzsch GmbH, Selb, Germany). 10 mg of sample was placed in an alumina crucible, and an empty crucible served as a control. These crucibles were heated from room temperature to 1100 °C at a heating rate of 10 °C / min in a flowing air stream.

[0061] Scanning electron microscopy (SEM): SEM photographs of the samples were obtained using a field-emission gun SEM microscope (FEG-SEM ΣIGMA, ZEISS NTS GmbH, Oberkochen, Germany). Powder samples were deposited on conductive tape attached to an SEM stub. The samples were observed in secondary electron acquisition mode without coating at low accelerating voltages (1–5 kV). Energy-dispersive X-ray spectra (SEM-EDS) were collected on the same samples at high accelerating voltages (15–20 kV).

[0062] Ion release in artificial saliva 200 mg of sample powder was dispersed in 10 mL of artificial saliva prepared as a modified Tani-Zucchi solution containing 20 mM KCl, 5.3 mM KSCN, 1.4 mM NaHPO, 15 mM NaHCO, and 10 mM lactic acid. The suspension was maintained at 37°C with shaking. At designated time points, 8 mL of the supernatant (separated from the solid phase by centrifugation at 7000 RPM for 5 minutes) was collected and ions were quantified using ICP-OES and a fluoride ion-selective electrode. The sample was then washed with 8 mL of fresh artificial saliva, and the suspension was again shaken at 37°C until the next time point.

[0063] Mineralization and ion release tests of SBF 37.5 mg of powder sample was dispersed in triplicate in 25 mL of simulated body fluid (SBF) prepared according to the method of Kokubo et al. [4]. The dispersions were kept at 37°C under horizontal shaking for the specified time (1, 3, 7, or 14 days). The solid and liquid were then separated by centrifugation (7000 RPM, 20°C, 5 minutes).

[0064] The supernatant was collected and analyzed for Ca, P, Si, and F by inductively coupled plasma optical emission spectrometry (ICP-OES) and fluoride ion-selective electrode (ISEF), respectively. ICP-OES was performed using an Agilent 5100 instrument (Agilent Technologies, Santa Clara, CA, USA), and the ISEF was performed using an Intellical ISEF121 electrode (Hach Lange, Loveland, CO, USA). Prior to ICP-OES analysis, the supernatant was diluted 1:3 with 1 wt% HNO3 aqueous solution. F analysis was performed according to the instrument manufacturer's instructions. Fluoride standard solutions were prepared from certified standards (1,000 ppm F certified standard, Sigma Aldrich, St. Louis, MO, USA). A standard calibration curve was obtained by analyzing the standard solutions on the day of preparation.

[0065] The pellets were washed with ultrapure water and freeze-dried, after which these dried materials were analyzed by PXRD, SEM, and SEM-EDS as described above.

[0066] Results and Discussion Morphological, structural, and compositional characterization of ACP / BG composite materials SEM micrographs of the ACP / BG composite material show BG microparticles (columnar microcrystals of 1 μm to 10 μm) surrounded by and immersed in a matrix of ACP nanoparticles (spherical nanoparticles with a diameter of approximately 50 nm) (Figure 1A-C). In composite materials with a high ACP:BG ratio, the relative abundance of BG microcrystals decreases, confirming that the composition of this material can be controlled. Low-magnification SEM micrographs show no separation between the ACP and BG. On the other hand, SEM micrographs of physical mixtures of ACP and BG show that the powder material is not homogeneous at the microscopic level, with particle separation present between pure BG or pure ACP microgranules (Figure 1D).

[0067] SEM micrographs of ACP / BGN and ACP / MBGN composite materials show the presence of both ACP nanoparticles (spherical nanoparticles with a diameter of approximately 50 nm) and BGN or MBGN nanoparticles (smooth or porous spherical nanoparticles with a diameter of 100–300 nm, respectively) (Figure 1E–G). The relative abundance of ACP nanoparticles decreases in composite materials with a lower ACP:BG ratio, confirming that composition control is possible in this case as well. Low-magnification SEM micrographs show that ACP and BGN / MBGN form deeply entangled composite materials, with both components uniformly distributed without forming separate aggregates. On the other hand, physical mixtures of ACP and BG are not uniform, exhibiting microgranules of pure BGN / MBGN or pure ACP (Figure 1H). While a mixture of MBGN and FACP particles can be observed in some granules, this mixing likely occurred only on the surface of the granules.

[0068] The actual weight composition of the samples is given in Table 1 and was calculated by comparing the weight of the BG added during synthesis with the weight of the product according to the following formula:

number

number

[0069] As can be observed, the actual composition is similar to the nominal composition, with the 20 / 80 MBGN sample containing more MBGN components (20 / 80), while the BG sample has a smaller excess of BG (40 / 60).

[0070] Table 1. Comparison of actual and nominal compositions of ACP / MBGN and ACP / BG complexes. JPEG2025542286000004.jpg39167

[0071] The SEM-EDS spectra of all ACP / MBGN composite materials show the presence of Ca, P, F, and Si. The relative abundances of these elements (Table 2) are consistent with the ACP:BG ratio. That is, as the BG content of the composite material increases, the Ca, P, and F contents decrease, while the Si content increases. SEM-EDS also allows us to estimate the actual ACP:BG ratio, which is close to the nominal value. Regarding the molar ratios (Table 3), the P / F molar ratio is similar for all composite materials and close to the ACP value (approximately 4), suggesting that the fluoride doping of ACP is not affected by the presence of BG during the precipitation of the composite materials. Meanwhile, the Ca / Si and P / Si ratios decrease as the MBGN content increases, as expected. The Ca / P molar ratio (1.82) of the 20 / 80 composite material is close to the Ca / P value of ACP (approximately 1.80), since ACP is the major component of the composite material. On the other hand, in composite materials with a high MBGN content, the MBGN also contains calcium, so the Ca / P ratio increases to approximately 2 to 4. As expected, the SEM-EDS spectra of the physical mixtures of ACP and MBGN vary significantly depending on the region sampled, ranging from a Si-rich composition in the BG granules to a CaP-rich composition in the ACP granules.

[0072] In the case of composite materials between ACP nanoparticles and MBGN nanoparticles, the SSA of the composite materials with ACP / MBGN ratios of 80:20 and 60:40 BET (163 ± 16 m respectively 2 g -1 and 178 ± 18 m 2 g -1 ) is the SSA of pure MBGN and pure ACP BET (319 m each 2 g -1 and 290 m 2 g -1 ) [3,5]. This confirms that the two types of nanoparticles are highly entangled in the ACP-rich composite material. Indeed, the high SSA of pure MBGN and pure ACP BET The values ​​are due to their extensive micro- and mesoporosity [3,5]. On the other hand, physical mixtures of ACP and MBGN show higher SSA than the corresponding composite materials. BET (220 m each 2 g -1 vs. 163 m 2 g -1 ) which is another evidence that simple mixing cannot achieve the same particle homogenization. For the sample with ACP / MBGN of 20 / 80, its SSA BET (284 ± 28 m 2 g -1 ) is a pure MBGN SSA BET is similar to

[0073] Table 2. Composition (wt%) of ACP / MBGN samples by SEM-EDS JPEG2025542286000005.jpg47165

[0074] Table 3. Composition (molar ratio) of ACP / MBGN samples determined by SEM-EDS JPEG2025542286000006.jpg47165

[0075] SEM-EDS spectra of all ACP / BG composite materials from commercially available micro-bioglass samples show the presence of Ca, P, F, and Si. The molar ratios of these materials (Table 4) are consistent with the ACP:BG ratio. That is, as the BG content of the composite increases, the Ca / Si and P / Si ratios decrease. Regarding molar ratios, as noted above, the P / F molar ratio is similar for all composite materials and close to that of ACP. However, because both ACP and BG contain Ca and P, this measurement and the Ca / P molar ratio cannot be easily interpreted. Even in this case, SEM-EDS spectra of physical mixtures of ACP and BG vary significantly depending on the region sampled, ranging from a Si-rich composition in BG granules to a CaP-rich composition in ACP granules.

[0076] Table 4. Composition (wt%) of ACP / BGN samples by SEM-EDS JPEG2025542286000007.jpg62165

[0077] The properties of the composite materials were investigated by powder X-ray diffraction (PXRD) and infrared spectroscopy (FT-IR). PXRD revealed no diffraction peaks, indicating that all ACP / BG composite materials were amorphous (Figure 2A, B). For the ACP / BGN and ACP / MBGN composite materials, two broad ridges, attributed to amorphous nanosilica and amorphous calcium phosphate, were observed at approximately 22° and 30°, respectively [6, 7]. The intensity ratio of the silica band to the ACP band was proportional to the ACP:BG ratio, and similar results have been observed for physical mixtures of ACP and BG. In the case of commercially available BG, the distinction is unclear, as 45S5 BG has a characteristic broad band between 30° and 35°, which is therefore partially overlapped with the ACP band (Figure 2A).

[0078] The FT-IR spectrum of the composite material further confirms its nature (Figure 2C,D). In particular, the major IR bands are due to the phosphate groups in the amorphous environment (1010, 960, and 555 cm, respectively). -1broad bands at v3, v1, v4PO4 modes) or silicate groups (1075, 800, and 450 cm, respectively) -1 The relative intensities of all these bands correlate with the ACP:BG ratio and shift accordingly. Also, as previously reported [5], the bands at 1400–1600 cm -1 Other bands were observed in the range of 1000 s, corresponding to carbonate ions (vCO3 mode) and citrate ions (vCOO mode) incorporated into the ACP, respectively. For physical mixtures of ACP and BG, the FT-IR spectra are not reproducible due to the heterogeneous composition of the materials. Furthermore, FT-IR spectroscopy of the ACP / BG composite material yields less clear results than ACP / BGN and ACP / MBGN because 45S5 bioglass contains both phosphate and silicate bands. However, the above findings are still clearly observable.

[0079] Ion release and calcification of SBF The primary tests to evaluate the remineralization ability of the ACP / BG composite material were (i) its ion release in acidic artificial saliva (modified Tani-Zucchi solution) that mimics the cariogenic oral environment, and (ii) its ability to induce HA crystallization upon contact with simulated body fluids.

[0080] Regarding ion release in artificial saliva, all composite materials showed release of Ca, P, Si, and F (Figure 3). This release was most intense (largest) in the first 2 hours, continued for up to 5 hours, and then gradually decreased, resulting in a slower release up to 24 hours. Comparison of the release curves showed that pure ACP only showed a large release of Ca, P, and F within 2 hours. Pure BG showed less intense release of Ca, P, and Si, which was dispersed over 24 hours.

[0081] For the ACP / BG composite material, the release was intermediate between that of pure ACP and pure BG and depended on the ACP:BG ratio. Specifically, the composite material with a high ACP content exhibited stronger Ca, P, and F release and weaker Si release. On the other hand, the behavior was reversed for the composite material with a high BG content (Figure 3A-D). This indicates that ion release can be tuned by adjusting the ACP:BG ratio. Most importantly, the composite material with a 60:40 ACP / BG ratio exhibited stronger Ca, P, and F release and weaker Si release compared to a physical mixture of ACP and BG at the same weight ratio. This clearly demonstrates that the preparation of this composite material can achieve superior ion release properties that cannot be achieved by simply mixing the two basic components.

[0082] For the ACP / MBGN composite material, the observed behavior is the same, but the distinction between the composite material, pure MBGN, and the physical mixture of ACP and MBGN is reduced and even attenuated (Figure 3E-G). This is due to the stronger ion release of the porous, nanoparticulate MBGN than the micro-MBGN. Notably, the F of the physical mixture of ACP and MBGN is - The release is higher than any of the composite materials and comparable to that of pure ACP, demonstrating that the composite materials allow tunable ion release, whereas simple mixing of the base materials does not.

[0083] Another important test to evaluate the remineralization potential of ACP / BG composite materials is their ability to induce HA crystallization upon contact with simulated body fluid (SBF), by simultaneously (i) crystallizing ACP into HA and (ii) precipitation of calcium and phosphate ions contained in SBF as new minerals through interaction with ACP / BG. In parallel with the release of Si in solution,

[0084] This test was carried out on the ACP / MBGN composite materials. The results of monitoring the ion concentrations in the liquid phase showed that, compared with the initial ion contents of SBF, Ca, P, and Mg gradually decreased in all ACP / MBGN composite materials, and all curves leveled off over time, indicating that precipitation of inorganic minerals occurred.

[0085] The amount of precipitated ions is directly proportional to the ACP:BG ratio, while Si release is inversely proportional. After 3 days of incubation, all of the phosphate in the solution is consumed, while Ca and Mg are only partially removed.

[0086] Compared to pure ACP, ACP clearly induces calcium phosphate precipitation immediately due to the seeding effect, showing the highest ion depletion even in a short time, whereas compared to pure MBGN, precipitation is shown to slow down over time, due to the first release of calcium and silicate from MBGN, followed by supersaturation, which induces the precipitation of calcium phosphate minerals.

[0087] Therefore, the behavior of the ACP / MBGN composite materials in SBF is controlled by the ACP:MBGN ratio. Observing the ion concentrations in SBF over time, the data for Ca, Mg, and P suggest that all composite materials induce the precipitation of calcium phosphate phases with Mg addition until all the P in solution is consumed, with the precipitation being faster for ACP and ACP-rich composite materials.

[0088] Comparison with physical mixtures of ACP and MBGN demonstrates that mixing the two components does not produce the same effect, as the precipitation of Ca, P, and Mg is less intense in the composite material and the release of Si is stronger. These analyses demonstrate that all composite materials induce the formation of a new Mg-doped calcium phosphate phase upon contact with SBF, and that the ACP component accelerates the mineralization process and enhances Mg and F doping, while the presence of MBGN results in sustained Si release. Furthermore, the rate of mineral formation can be controlled by adjusting the ACP:MBGN ratio.

[0089] We analyzed the materials after immersion in SBF using SEM (Figure 4). In the ACP / MBGN composite material, the morphology of the MBGN particles remained unchanged, but the spherical ACP nanoparticles were observed to have transformed into small, needle-like nanocrystals (Figure 4A–C). These crystals were less than 20 nm wide and less than 100 nm long and were present throughout the sample. This morphology was similar to that of the pure ACP control sample (Figure 4E). In contrast, in the pure MBGN sample, thick needle-like crystals formed on the MBGN particles and grew over time as clusters of elongated crystals approximately 200–400 nm long and approximately 50 nm wide (Figure 4D). Many studies have shown that immersion in SBF induces the formation of HA, while ACP converts it to HA. Therefore, the new crystals observed in this composite material are HA nanocrystals formed by ion release and reprecipitation by both MBGN and ACP (thick needle-like crystals) and direct conversion of ACP (thin needle-like crystals). In this composite material, the ACP component controls the rate of HA formation, inducing the formation of new HA nanocrystals in proportion to the ACP:MBGN weight ratio.

[0090] 1. Cochrane, NJ et al., A novel approach to promoting tooth enamel remineralization. Journal of Dental Research, 2010. 89(11): p. 1187-1197. 2. Neel, EAA et al., Dynamic demineralization-remineralization in teeth and bone. International Journal of Nanomedicine, 2016. 11: pp. 4743-4763. 3. Zheng, K. et al., Towards highly dispersed mesoporous bioactive glass nanoparticles containing high concentrations of copper: Using Cu / ascorbic acid complex as a precursor. Frontiers in Chemistry, 2019. 7: p. 497. 4. Kokubo, T. and H. Takadama, How useful is SBF for predicting in vivo bone bioactivity? Biomaterials, 2006. 27(15): pp. 2907-2915. 5. Iafisco, M. et al., Fluoridated amorphous calcium phosphate nanoparticles: a promising biomimetic material for tooth remineralization. Scientific Reports, 2018. 8(1): p. 17016. 6. Bari, A. et al., Copper-containing mesoporous bioactive glass nanoparticles as multifunctional agents for bone regeneration. Acta Biomaterialia, 2017. 55: p. 493-504. 7. Combes, C. and C. Rey, Amorphous calcium phosphates: synthesis, properties, and applications in biomaterials. Acta Biomaterialia, 2010. 6(9): p. 3362-3378. 8. Aguiar, H. et al., Structural study of sol-gel silicate glasses by IR and Raman spectroscopy. Journal of Non-Crystalline Solids, 2009. 355(8): p. 475-480. 9. Antonakos, A., E. Liarokapis, and T. Leventouri, Micro-Raman and FTIR studies of synthetic and natural apatites. Biomaterials, 2007. 28(19): p. 3043-3054.

Claims

1. A composite material comprising or consisting of particles of amorphous calcium phosphate and particles of bioactive glass (ACP / BG), wherein the BG particles are surrounded by and immersed in a matrix of ACP particles, thereby forming a homogeneous, intimately intertwined composite material.

2. The composite of claim 1 , wherein the BG particles are macroparticles, microparticles, or nanoparticles, preferably porous or mesoporous.

3. 3. The composite of claim 1 or 2, wherein the ACP particles are doped with one or more ions of F, Zn, Mg, Sr, Na, K, Cu, Fe, Cl, and Ag.

4. A method for preparing the complex according to any one of claims 1 to 3, comprising: a) mixing an aqueous solution (Solution A) containing calcium salt and / or calcium hydroxide, BG particles, and sodium or potassium citrate and / or citric acid with a solution (Solution B) containing phosphate and / or phosphoric acid, and carbonate and / or carbonic acid; b) stirring the mixture for at least 3 seconds to form a precipitate; and c) collecting and drying the precipitate A method comprising:

5. 5. The method of claim 4, wherein in step c) the precipitate is dried by freeze-drying.

6. The method according to claim 4 or 5, wherein, prior to step a), solution A is sonicated to disperse the BG particles and the pH of solution B is made basic, greater than 9.

7. 7. The method according to claim 4, wherein solution A contains zinc, strontium, iron, silver, copper, sodium, potassium, and / or magnesium ions as dopants for ACP; and solution B contains fluorine and / or chlorine ions as dopants for ACP.

8. The composite material according to any one of claims 1 to 3 for dental use.

9. 9. A composite material for use according to claim 8 as a remineralisation agent.

10. 10. A composite material for use according to claim 9 for preventing tooth demineralisation and / or promoting tooth remineralisation.

11. 9. A composite material for use according to claim 8 for preventing and / or treating dental caries and cavities.

12. 9. A composite material for use according to claim 8 for reducing dentin hypersensitivity by occluding dentin tubules with said composite.

13. 4. The composite material according to any one of claims 1 to 3 for use as a filler for bone defects or in the form of a scaffold or as a coating for orthopedic implants for bone regeneration.

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