EP4638356A1

EP4638356A1 - Composite material based on bioactive glass and amorphous calcium phosphate, and the process for producing the same

Info

Publication number: EP4638356A1
Application number: EP23837426.8A
Authority: EP
Inventors: Lorenzo DEGLI ESPOSTI; Michele Iafisco; Aldo R. BOCCACCINI; Kai ZHENG
Original assignee: Friedrich Alexander Universitaet Erlangen Nuernberg; Consiglio Nazionale delle Richerche CNR
Current assignee: Friedrich Alexander Universitaet Erlangen Nuernberg; Consiglio Nazionale delle Richerche CNR
Priority date: 2022-12-22
Filing date: 2023-12-20
Publication date: 2025-10-29
Also published as: JP2025542286A; WO2024134524A1; IT202200026496A1; CN120584086A; MX2025007406A

Abstract

The invention relates to an amorphous calcium phosphate/bioactive glass composite material (ACP/BG). This composite material possesses both the short-time ion release capability of ACP as well as the long-time ion release of BG and thus (i) efficiently remineralizes damaged enamel, and (ii) induces the formation of a new HA layer onto enamel and dentin surface. The ACP/BG composite material is obtained by embedding BG particles in a matrix of ACP nanoparticles in order to maximize homogenization between the two components and to produce a composite with high uniformity from the macro- to the nano-scale. This has been achieved with a process that includes a step of precipitating ACP onto BG particles. The BG particles can be nano-, micro-, or macro-particles. The particles can also be porous or mesoporous The invention relates also to the use of the composite material of the invention for use in the prevention of dental demineralization and to boost remineralization. In other words, the composite material here disclosed is used as remineralizing agent thanks to its ability to restore the structure and preserve the mechanical properties of dental hard tissues by releasing Ca2+ and PO4 3− ions, thereby mimicking the spontaneous remineralization process induced by saliva. The composite material can also be used for bone regeneration.

Description

Composite material based on bioactive glass and amorphous calcium phosphate, and the process for producing the same

DESCRIPTION

FIELD OF THE INVENTION

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

BACKGROUND OF THE INVENTION

According to the Global Burden of Disease study of 2017, dental caries is the most widespread non-communicable disease which affects both industrialized and developing countries. The disease results from a prevalence of acidogenic bacteria in the biofilm that permanently colonizes any oral surface. These cariogenic bacteria produce organic acids, and the consequence is a lowering of the pH in the microenvironment of teeth surfaces (enamel and dentine), which triggers the dissolution of their mineral component, hydroxyapatite (HA, Caio(P04)e(OH)2). HA dissolution is referred to as the “demineralization process" and leads to the formation of dental cavities. Demineralization is a reversible process if the damaged tissues are exposed to an oral environment that favors the opposite process, “remineralization". Demineralized enamel is naturally remineralized by the epitaxial growth of residual HA crystals that act as nucleation sites, as saliva provides a supersaturated environment rich of Ca²⁺ and PO4³" ions. However, remineralization of enamel by saliva alone is seldom achieved. Therefore, it is required the use of an external source of Ca²⁺ and PO4³" ions to increase ion supersaturation to efficiently prevent demineralization and boost remineralization. This is the aim of remineralizing agents, which restore the structure and preserve the mechanical properties of dental hard tissues by releasing Ca²⁺ and PO4³" ions, [1J mimicking the spontaneous remineralization process induced by saliva. Currently there is a high interest in remineralizing agents because modern dentistry is increasingly oriented towards early prevention of tooth decay rather than invasive restorative therapy. Indeed, the best preventive approach for dental caries is to stop their formation and progression by promoting the remineralization of tooth surfaces.

For this aim [2] various forms of synthetic calcium phosphates have been proposed, as these materials mimic the composition and structure of the mineral phase of teeth and thus are ideal for releasing Ca²⁺ and PO4³" ions. The most used synthetic calcium phosphates for dental remineralization are HA, fluoro-hydroxyapatite (FHA), and amorphous calcium phosphate (ACP). These calcium phosphates agents can be either added to restorative materials or be directly applied onto the tooth surface.

Among these calcium phosphates agents, ACP is particularly appealing in dentistry due to its ability to release a higher amount of ions in comparison to crystalline calcium phosphates phases. Several ACP products are actually marketed for dental remineralization in formulations that are directly applied on tooth surface (i.e. tooth mousses) having a good level of literature evidence for this use (systematic reviews). ACP is an unstable material that rapidly transforms into more thermodynamically stable crystalline phases (e.g., HA) in solution or in dry state by reacting with atmospheric water. Due to this instability, use and handling of ACP is difficult, and several additives were studied to stabilize ACP. In this regard, W02020/002517 and WO2016/012452 describe a fluoride-doped amorphous calcium phosphate (F-ACP) stabilized by citrate ions that in aqueous solution gives burst release of Ca²⁺, PO4³; and F’ ions, is able to crystallize into HA when in contact with enamel and dentine, inhibits cariogenic biofilm formation, and shows a strong remineralizing activity. The known F-ACP has been proved, therefore, to be an excellent material for dental remineralization. The main limit of all ACP materials (either F-ACP or commercial ACP products) is that their burst release lasts for a short timeframe, and thus several applications of the product are needed for a complete remineralization.

The other prominent class of materials that are currently used as remineralizing agents are bioactive glasses (BGs). BGs are well-known materials for hard tissue regeneration, as they are bioactive (HA-forming ability), biocompatible, osteoconductive, osteoinductive, and gives a controlled release of bioactive ions in the surrounding environment. In addition, bioactive glass nanoparticles (BGN) have attracted increasing attention due to their small size and large specific surface area which consequently lead to unique features such as higher bioactivity and higher loading and release of ions, drugs, and other biomolecules. An advantage of BG and BGN materials is that they can be fabricated with a wide range of ionic substitutions, allowing to release many bioactive ions over time. As consequence of their controlled ion release capacity, BG have found a successful application as remineralizing agents. However, a serious drawback that hinders this application is that BG cannot release a large amount of Ca²⁺ and PO4³" ions and is not able to perform completely its remineralization function to form HA because it is diluted away by salivary action in a short time before it attaches to dental surfaces. Therefore, by considering the state of the art on remineralizing agents there is an unmet need of a material that releases remineralizing (Ca²⁺, PO4³") and bioactive (e.g., F; Si⁴⁺, Sr²⁺, Zn²⁺, Mg²⁺) ions both in the short as well as in the long timeframe, and at the same time continuously induces the formation of new HA crystallites onto depleted enamel and dentine.

SUMMARY OF THE INVENTION

The present invention relates to an amorphous calcium phosphate/bioactive glass composite material (ACP/BG). This composite material possesses both the short-time ion release capability of ACP as well as the long-time ion release of BG and thus (i) efficiently remineralizes damaged enamel, and (ii) induces the formation of a new HA layer onto enamel and dentin surface. In detail, ACP component has a twofold action: (i) gives a burst ion release for initial HA formation (Ca²⁺, PO4³ F- as well as Sr²⁺, Mg²⁺, Zn²⁺) and (ii) acts as nucleation site for HA growth by attaching onto enamel and dentine surface, while the ion-doped BG component provides for a sustained ion release of remineralizing and biologically active ions (e.g., Ca²⁺, PO4³", F; Si⁴⁺, Sr²⁺, Zn²⁺ Mg²⁺), thereby generating a continuous remineralization.

The ACP/BG composite material is obtained by embedding BG particles in a matrix of ACP nanoparticles in order to maximize homogenization between the two components and to produce a composite with high uniformity from the macro- to the nano-scale. This has been achieved with a process that includes a step of precipitating ACP onto BG particles. The BG particles can be nano-, micro-, or macro-particles. The particles can also be porous or mesoporous

The invention relates also to the use of the composite material of the invention for use in the prevention of dental demineralization and to boost remineralization. In other words, the composite material here disclosed is used as remineralizing agent thanks to its ability to restore the structure and preserve the mechanical properties of dental hard tissues by releasing Ca²⁺ and PO4³" ions, thereby mimicking the spontaneous remineralization process induced by saliva.

The composite material can also be used for bone regeneration.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. (A-H) SEM micrographs of (A) ACP/BG 80/20 composite, (B) 60/40 composite, (C) 40/60 composite, and (D) ACP+BG physical mixture, (E) ACP/MBGN 80/20 composite, (F) 60/40 composite, (G) 40/60 composite, and (H) ACP+MBGN physical mixture. Light grey arrows point at BG or MBGN particles, black arrows at ACP particles.

Figure 2. PXRD patterns of (A) ACP/BG composites, and (B) ACP/MBGN composites. FT-IR spectra of (C) ACP/BG composites, and (D) ACP/MBGN composites.

Figure 3. (A-G) Cumulative (A) Ca, (C) P, (E) Si, and (G) F ion release of ACP/BG composites and physical mixture, and cumulative (B) Ca, (D) P, (F) Si ion, and (H) F ion release of ACP/MBGN composites and physical mixture.

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

DETAILED DESCRIPTION OF THE INVENTION

The composite material of the invention comprises or consists of bioactive glass particles and amorphous calcium phosphate particles as an intimate and homogenous mixture obtained by precipitating ACP onto BG particles.

BG particles are chosen among macroscopic particles, micrometric particles or nanoparticles. Each of them can be microporous or mesoporous. BG nanoparticles are defined as BGN and mesoporous BG nanoparticles are defined as MBGN.

Compositions of macro and micrometric BG particles typically contain SiC>2, CaO, Na2<D and P2O5 in different ratios. Other components, e.g., ZnO, CuO, SrO, can also be incorporated in BG compositions.

Compositions of BGN and MBGN typically contain SiO2 and CaO in different ratios. For instance, BGN are made of 90% SiO2 and 10% CaO, while MBGN are made of 10% CaO and 90% SiO2. Other components, e.g., ZnO, CuO, SrO, can also be incorporated into BG nanoparticles and MBGN

Preferably, ACP is in the form of nanoparticles.

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

The composite material comprises ACP in an amount ranging between 1 % wt and 99% wt and BG in an amount ranging from 99% wt and 1 % wt.

In another embodiment, the composite material comprises ACP in amount ranging between 20% and 80%, or 20% and 60%, or 20% and 40% and BG in an amount ranging from 80% and 20%, or 60% and 20%, or 40% and 20%.

The two components are intimately and homogenously intertwined together to yield the superior properties demonstrated by the composite material with respect to a simple mixture of the two components ACP and BG.

The composite material bypasses the intrinsic disadvantages that its constituting components have, such as poor ion release on the short timeframe, limited apatite formation, and no fluoride release for BG, and no sustained release and low stability for ACP.

The composite material is obtained with a process that comprises the following steps: a) Mixing an aqueous solution comprising a calcium salt and/or calcium hydroxide, BG particles and a sodium or potassium citrate salt and/or citric acid (solution A) with a solution comprising a phosphate salt and/or phosphoric acid and a carbonate salt and/or carbonic acid (solution B); b) Stirring the mixture for at least 3 seconds to allow the formation of a precipitate; c) Collecting and drying the precipitate.

The calcium salt is chosen among calcium chloride, calcium nitrate, calcium acetate, calcium lactate, calcium oxalate, calcium citrate, and calcium sulfate,

The sodium or potassium citrate salt is chosen among sodium citrate tribasic, sodium citrate dibasic, sodium citrate monobasic, potassium citrate tribasic, potassium citrate dibasic, and potassium citrate monobasic.

The phosphate salt is chosen among sodium phosphate, sodium hydrogen phosphate, sodium dihydrogen phosphate, or potassium phosphate, potassium hydrogen phosphate, potassium dihydrogen phosphate, or ammonium phosphate, ammonium hydrogen phosphate, ammonium dihydrogen phosphate.

The carbonate salt is chosen among sodium carbonate, sodium hydrogen carbonate, sodium dihydrogen carbonate, or potassium carbonate, potassium hydrogen carbonate, potassium dihydrogen carbonate, or ammonium carbonate, ammonium hydrogen carbonate, ammonium dihydrogen carbonate.

The mixture is stirred in step b) for a time of 3 sec-1 h, preferably 10 sec-60 sec.

The precipitate is collected for example by centrifugation or by filtration or by vacuum filtration or by decantation and then exsiccated for example by freeze-drying, by heating or spray drying. BG particles, which can be BG macro or microparticles, BGN or MBGN are prepared with methods known in the art or are commercially available.

Before step a) solution A can be optionally sonicated to disperse the BG particles and the pH of solution B can be optionally brought to a basic pH of above 9.

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

Solution A can optionally include zinc, strontium, iron, silver, copper, sodium, potassium, and/or magnesium ions that are doping agents for ACP.

Solution B can optionally include fluorine, and/or chlorine ions that are doping agents for ACP.

The mixing step a) is preferably performed at a temperature between 1-30°C, or 4-25°C, or 20-25°C.

The composite material obtained by the process of the invention has a structure in which the BG particles are surrounded and immersed in a matrix of ACP nanoparticles. There is no segregation between ACP and BG and the mixture is homogenous and intimately mixed, forming a deeply-intertwined composite.

The relative abundance of BG microparticles decreases in the composite with higher ACP:BG ratios, confirming that it is possible to control the composition of the composite material.

A comparison between the composite of the invention and a physical mixture of ACP and BG show that the physical mixture is not homogenous, presenting micrometric granules of pure BGN/MBGN or pure ACP.

The composite of the invention has shown a good release of Ca, P, Si and optionally F, Zn and/or Mg, if doped with one or more of those ions. The release is more intense for a first period of at least 2 h and remains sustained for a second period of up to 5 h. Afterwards the release gradually slows down continuing as a slow release up to 24 hours.

The release curves have been compared to pure ACP, which shows only a burst release of Ca, P, and F in less than 2 h, and to pure BG, whose release of Ca, P, and Si is less intense and is spread across the 24 h timeframe.

In the case of ACP/BG composites, their release is intermediate between the ones of pure ACP and pure BG and depends on ACP:BG ratio, i.e. ACP-rich composites have a more intense Ca, P, and F release and less intense Si release, while in BG-rich composites the behavior is the opposite. This proves that ion release can be modulated by adjusting the ACP:BG ratio. The most important finding is that ACP/BG composite has a stronger Ca, P, and F release and a weaker Si release than the ACP+BG physical mixture with identical weight ratio. This is the definitive proof that the composite preparation gives better ion-releasing properties which cannot be achieved by only mixing the two base ingredients.

In the case of ACP/MBGN composites, the observed behavior is the same, but the differences are more attenuated with less distinction between composites, pure MBGN, and ACP+MBGN physical mixture.

The tests performed by the applicant demonstrate that the composite of the invention can be used for the prevention of dental demineralization and to boost remineralization. These effects help to prevent and treat dental caries and cavities.

The composite of the invention can also be used to achieve dentin desensitization by occluding the dentinal tubules.

Another use of the composite of the invention is for bone regeneration.

The composite material can also be used for bone regeneration. Owing to its outstanding remineralization and ion release capacity, the composite material can be used as bone defect filler to accelerate formation of HA and stimulate osteogenesis and angiogenesis. The composite material can also be used to fabricate orthopedic implant coating or bone tissue engineering scaffolds for bone repair and regeneration. The fast release of Ca²⁺ and PO4³" ions from ACP facilitates bone bonding while sustained release of biologically active ions from BGs ensures favorable biological responses for bone regeneration.

The invention refers also to a method to prevent dental demineralization and/or to boost dental remineralization which includes a step of applying the composite material on dental hard tissues.

Material and Methods

Materials and precursors

Reagents needed for composite preparation:

Calcium chloride dihydrate (CaCl2-2H2O, >99.0% pure), hydrochloric acid (HCI, >37.0% pure), sodium citrate tribasic dihydrate (Na3(C6H5O7) 2H2O, >99.0% pure, hereafter called sodium citrate), sodium phosphate dibasic dihydrate (Na2HPO4-2H2O, >99.0% pure), sodium carbonate monohydrate (Na2CO3-2H2O, >99.0% pure), sodium fluoride (NaF, >99.0% pure), MBGN, BGN, BG. MBGN were synthesized and characterized as reported by Zheng et al. [3] using a microemulsion-based sol-gel method. In detail, in a typical process, 0.56 g of cetrimonium bromide (CTAB, > 97%, Sigma-Aldrich) was dissolved in 26 mL of deionized water under stirring. 8 mL of ethyl acetate (>99.8%, Sigma-Aldrich) was added when CTAB was dissolved entirely. 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 (>99.4%, VWR) were added sequentially at an interval of 30 min followed by further 4 h of reaction under stirring. The formed colloids were then collected by centrifugation at 7197 ref for 20 min and washed twice with deionized water and once with ethanol (96%, VWR). The collected deposits were then dried at 60°C overnight before calcination at 700°C for 4 h with a heating rate of 2°C/min.

BGN nanoparticles were synthesized using a modified Stober method (Zheng K, et al. Timing of calcium nitrate addition affects morphology, dispersity and composition of bioactive glass nanoparticles[J], RSC advances, 2016, 6(97): 95101 -95111 ). In a typical synthesis process, a solution (A) composed of 2.25 mL of tetraethyl orthosilicate (TEOS, 98%, Sigma-Aldrich) and in 25 mL of ethanol (96% VWR) was mixed with Solution B that 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 leaving the reaction to proceed for 30 min, 1.45 g of calcium nitrate tetrahydrate was added. The mixtures were allowed to react for a further 90 min before collection by centrifugation at 7197 ref for 25 min. The obtained particles were dispersed and washed twice with deionized water and once with ethanol. The collected particles were subsequently dried at 60 °C overnight before calcination at 700 °C for 2 h with a heating rate of 2 °C/min.

Commercial “45S5-type” BG microparticles were acquired from Schott glass - commercial name: Schott Vitryxx Bioactive Glass Powder, size SM4.0, material MD01

ACP/BG Composite preparation

ACP/BG composites were prepared by mixing at room temperature equal volumes of two aqueous solutions, consisting of (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 give a final ACP:BG weight ratio of 80:20, 60:40, or 40:60 (where X = 2 mg/mL for ACP/BG 80/20, 6 mg/mL for ACP/BG 60/40, and 18 mg/mL for ACP/BG 40/60). Before mixing, solution (A) was sonicated at 20% amplitude for 3 min with a pulsation of 5 s under ice cooling employing a Vibracell VCX 500 tip sonicator (SONICS, Newtown, CT, USA) to disperse BG, while the pH of solution (B) was brought to 9.5 with HCI 37 wt.%. After mixing, the precipitate was left to stir for at least 30 s at room temperature and afterwards the particles were collected by centrifugation (7000 RPM, 5 min, 4 °C) and repeatedly washed with ultrapure water. Finally, the materials were freeze-dried for 24h.

Control composite preparation

The 60:40 wt.% ACP+BG physical mixture was prepared by hand mixing together 240 mg of ACP powder and 160 mg of BG powder (either BG, BGN, or MBGN) until complete homogeneity was achieved.

Chemical, morphological and structural characterization

Powder X-ray diffraction (PXRD): PXRD patterns of the samples were recorded on a D8 Advance diffractometer (Broker, Karlsruhe, Germany) using Cu Ka radiation generated at 40 kV and 40 mA. PXRD patterns were collected in the 10 - 60° 29 range with a step size of 0.02 degree and a collection time of 0.5 s.

Fourier transform infrared spectroscopy (FT-IR): FT-IR spectra were collected on a Nicolet 5700 spectrometer equipped with an ATR iD7 accessory (Thermo Fisher Scientific Inc., Waltham, MA, USA). Spectra were collected in ATR mode by accumulation of 32 scans in the range between 4000 to 400 cm^-1 with a resolution of 2 cm^-1.

Specific Surface Area analysis (SSABET): The specific surface area of the samples was measured through N2 gas adsorption method using a Sorpty 1750 instrument (Carlo Erba, Milan Italy) and Brunauer-Emmett-Teller (BET) approach.

Thermal characterization: Thermogravimetry analysis (TGA) of the samples was performed with a STA 449F3 Jupiter instrument (Netzsch GmbH, Selb, Germany). 10 mg of the samples were placed in an alumina crucible and an empty crucible was used as a reference. The crucibles were heated from room temperature to 1100 °C with a heating rate of 10 °C/min under air flow.

Scanning electron microscopy (SEM): SEM micrographs of the samples were acquired with field-emission gun SEM microscope (FEG-SEM ZIGMA, ZEISS NTS Gmbh, Oberkochen, Germany). Powdered samples were deposited onto a conductive tape mounted on a SEM stub. The samples were observed in secondary electrons acquisition mode without coating at low acceleration voltage (1-5 kV). Energy dispersive X-ray spectra (SEM-EDS) were collected on the same samples at higher acceleration voltage (15-20 kV).

Ion release in artificial saliva

200mg of sample powders were dispersed into 10mL of artificial saliva prepared as modified Tani-Zucchi solution containing KCI 20mM, KSCN 5.3mM, Na2HPO4 1.4mM, NaHCOs 15mM, and lactic acid 10mM. The suspension was maintained at 37 °C under shaking. At scheduled times 8m L of the supernatant (that was separated from the solid phase by centrifugation at 7000 rpm for 5m in) was removed for ions quantification by ICP-OES and fluoride ion selective electrode. After that, samples were rinsed with 8m L of fresh artificial saliva, and the suspension was again shacked at 37 °C until the next time point.

SBF mineralization and ion release test

37.5 mg of the powdered samples were dispersed in triplicate in 25 mL of simulated body fluid (SBF) prepared according to Kokubo et al. [4], The dispersions were kept at 37°C under horizontal shaking for a determined time (1 , 3, 7, or 14 days). Afterward, the solid was separated from the liquid by centrifugation (7000 RPM, 5 min, 20 °C).

The supernatant was collected and analyzed by inductively-coupled plasma optical emission spectrometry (ICP-OES) and by ion-selective electrode for fluoride (ISEF) for quantification of Ca, P, Si, and F, respectively. ICP-OES was performed with an Agilent 5100 instrument (Agilent Technologies, Santa Clara, CA, USA), while ISEF was an Intellical ISEF121 electrode (Hach Lange, Loveland, CO, USA). Before ICP-OES analysis, the supernatants were diluted 1 :3 with a 1 wt. % HNOs aqueous solution. F analyses were performed as suggested by instrument manufacturer. Standard fluoride solutions were prepared from a certified standard (1 ,000 ppm F certified standard, Sigma Aldrich, St. Luis, MO, United States); the calibration curve was then obtained by analyzing the standard solutions on the same day they were prepared.

The pellet was washed with ultrapure water and freeze-dried. Afterward, the dried materials were analyzed by PXRD, SEM, and SEM-EDS as reported above.

Results and discussion Morphological, structural, and compositional characterization of ACP/BG composites

SEM micrographs of the ACP/BG composites show that BG micrometric particles (prismatic microcrystals ranging from 1 pm to 10 pm) are surrounded and immersed in a matrix of ACP nanoparticles (round-shaped nanoparticles with diameter of ca. 50 nm) (Figure 1A-C). The relative abundance of BG microcrystals decreases in the composites with higher ACP:BG ratios, confirming that is possible to control the composition of the materials. Low-magnification SEM micrographs show that there is no segregation between ACP and BG. On the contrary, SEM micrographs of ACP+BG physical mixtures shows that at microscopic level the powdered material is not homogeneous, and there is particle separation between micrometric granules of pure BG or pure ACP (Figure 1 D).

SEM micrographs of ACP/BGN and ACP/MBGN composites show both the presence of ACP nanoparticles (round-shaped nanoparticles with diameter of ca. 50 nm) as well as BGN or MBGN nanoparticles (respectively smooth or porous rounded nanoparticles with a diameter between 100 and 300 nm) (Figure 1 E-G). The relative abundance of ACP nanoparticles decreases in the composites with lower ACP:BG ratios, confirming that also in this case is possible to control the composition. Low-magnification SEM micrographs show that ACP and BGN/MBGN have formed a deeply-intertwined composite, and both components are homogeneously distributed without forming separated aggregates. On the other hand, ACP+BG physical mixtures are not homogeneous, presenting micrometric granules of pure BGN/MBGN or pure ACP (Figure 1 H). In some granules it is possible to observe a mixture of MBGN and FACP particles, but it is likely that the mixing occurred only on the surface of granules.

The actual weight composition of the samples is reported in Table 1 and was calculated by comparing the weight of BG added during synthesis and weight of the product according to the formulas: 1.00 used C wt.% content = - : - - — ; — ^: - * 100 product wei^nt

It can be observed that actual compositions are similar to the nominal ones, although that with 20/80 sample for MBGN is rich in MBGN component (20/80) while with BG there is less excess of BG (40/60).

Table 1. Actual composition of the ACP/MBGN and ACP/BG composites in comparison to nominal one.

Actual

Actual ACP:BG ratio

Sample ACP:MBGN Sample

(wt.%) ratio (wt.%)

ACP/MBGN 80/20 85/15 ± 5 ACP/BG 80/20 80/20 ± 5

ACP/MBGN 60/40 55/45 ± 5 ACP/BG 60/40 60/40 ± 5

ACP/MBGN 20/80 20/80 ± 5 ACP/BG 20/80 40/60 ± 5

ACP+BG phys.

ACP+MBGN phys. mix 60/40 ± 5 60/40 ± 5 mix

SEM-EDS spectra for all ACP/MBGN composites show the presence of Ca, P, F, and Si. The relative abundance of these elements (Table 2) is in agreement with ACP:BG ratios, i.e. Ca, P, F content decreases and Si content increases when the composites are more rich in BG. Also with SEM-EDS is possible to estimate the actual ACP:BG ratios, which are close to the nominal ones. Regarding molar ratios (Table 3), P/F molar ratio is similar for all the composites and close to ACP value (ca. 4), suggesting that fluoride doping of ACP is not influenced by the presence of BG during composite precipitation. On the other hand, Ca/Si and P/Si ratios decrease when MBGN is increased, as expected. Ca/P molar ratio of 20/80 composite (1.82) is close to ACP Ca/P value (ca. 1.80) as ACP is the dominant component in the composite, while in composites with higher MBGN content the Ca/P ratio increases to ca. 2-4 because also MBGN contains calcium. As expected, the SEM-EDS spectra of ACP+MBGN physical mixtures varies greatly depending on the sampled region, passing from a Si-rich composition for BG granules to a CaP-rich composition for ACP ones.

In the case of composites between nanoparticles (ACP/MBGN), the SSABET of ACP/MBGN 80:20 and 60:40 composites (163 ± 16 and 178 ± 18 m² g^-1, respectively) are significantly lower than both SSABET of pure MBGN and of pure ACP (319 m² g^_1 and 290 m² g^_1, respectively) [3, 5], This confirms that the two nanoparticle types are highly intertwined in ACP-rich composites. Indeed, the high SSABET values of pure MBGN and ACP are due to their extensive micro- and meso-porosities [3, 5], Differently, the ACP+MBGN physical mixture has a higher SSABET than the corresponding composite (220 vs 163 m² g’¹, respectively), which is another proof that the same particle homogenization cannot be achieved through simple mixing. In the case of ACP/MBGN 20/80 sample its SSABET (284 ± 28 m² g^-1) is similar to the SSABET of pure MBGN.

Table 2. Composition (wt.%) of the ACP/MBGN samples by SEM-EDS.

Ca P Si F SSABET (m²

Sample (wt%) (wt%) (wt%) (wt%) g-¹)

ACP/MBGN 26.3 ±

11.2 ± 0.3 6.6 ± 1.0 2.2 ± 0.1 178 ± 18

80/20 0.6

ACP/MBGN 18.4 ±

6.9 ± 0.6 22.0 ± 2.2 1.2 ± 0.2 163 ± 16

60/40 1.3

ACP/MBGN 10.1 ±

2.2 ± 0.8 39.1 ± 2.7 0.4 ± 0.1 284 ± 28

20/80 1 .4

ACP+MBGN n.a. n.a. n.a. n.a. 220 ± 22 phys. mix

ACP (reference) 31.9 ±

13.1 ± 0.3 0 1.5 ± 0.1 290 ± 29

[5] 0.8

Table 3. Composition (molar ratios) of the ACP/MBGN samples by SEM-EDS.

Ca/Si P/Si Ca/P

Sample P/F (mol)

(mol) (mol) (mol)

ACP/MBGN 80/20 2.9 ± 0.5 1.6 ± 0.3 1.82 ± 0.03 3.1 ± 0.2

ACP/MBGN 60/40 0.6 ± 0.1 0.4 ± 0.1 2.06 ± 0.06 3.5 ± 0.2

ACP/MBGN 20/80 0.18 ± 0.04 0.05 ± 0.02 3.80 ± 0.80 3.2 ± 0.1

ACP+MBGN phys. n.a. n.a. n.a. n.a. mix

ACP (reference) 0 0 « 1.80 ~ 4

SEM-EDS spectra for all ACP/BG composites with commercial micrometric bioglass show the presence of Ca, P, F, and Si. The molar ratio of these materials (Table 4) is in agreement with ACP:BG ratios, i.e. Ca/Si and P/Si decrease when the composites are more rich in BG. Regarding molar ratios, as above P/F molar ratio is similar for all the composites and close to ACP value, but this measure as well as Ca/P molar ratio cannot be interpreted easily since both ACP and BG contain Ca and P. Also in this case the SEM-EDS spectra of ACP+BG physical mixtures varies greatly depending on the sampled region, passing from a Si-rich composition for BG granules to a CaP-rich composition for ACP ones.

Table 4. Composition (wt.%) of the ACP/BGN samples by SEM-EDS.

Ca/Si P/Si Ca/P SSABET (m²

Sample P/F (mol)

(mol) (mol) (mol) g-¹)

6.9 ±

ACP/BG 80/20 4.2 ± 1.4 1.64 ± 0.05 2.4 ± 0.7 //

2.2

4.9 ±

ACP/BG 60/40 3.0 ± 1.1 1.61 ± 0.06 3.2 ± 0.9 //

1.9

1.6 ±

ACP/BG 20/80 0.9 ± 0.2 1.75 ± 0.01 2.9 ± 0.3 //

0.3

ACP+BG phys. n.a. n.a. n.a. n.a. // mix

ACP (reference) 0 0 « 1.80 « 4 290 ± 29

45S5 glass

« 0.8 « 0.06 « 13 0 Not reported

(reference)

45S5 glass 0.02 ±

0 ± 0 0 ± 0 0 ± 0 //

(experimental) 0.03

The nature of the composites was investigated by powder X-Ray diffraction (PXRD) and infrared spectroscopy (FT-IR). PXRD shows that all ACP/BG composites are amorphous, as no diffraction peaks can be observed (Figure 2A,B). In the case of ACP/BGN and ACP/MBGN composites, two broad humps attributed to amorphous nano-silica and amorphous calcium phosphate are observed at ca. 22° and 30°, respectively [6, 7], The intensity ratio between silica band and ACP band is proportional to ACP:BG ratio, and a similar result is observed also for ACP+BG physical mixture. For commercial BG the distinction is less clear as 45S5 BG has its characteristic broad band between 30° and 35°, so it is partially superimposed with ACP band (Figure 2A).

The FT-IR spectra of the composites gives a further confirmation about their nature (Figure 2C,D). In detail, the main IR bands correspond either to phosphate groups in an amorphous environment (vs, vi, and V4PO4 modes as broad bands at 1010, 960, and 555 cm^-1, respectively) or silicate groups (Si-O-Si stretching, bending, and rocking modes at 1075, 800, and 450 cm^-1, respectively) [8, 9], The relative intensity of all these bands is correlated to the ACP:BG ratio and shift accordingly. In addition, other bands can be observed in the range 1400-1600 cm’¹ that correspond respectively to carbonate (V2CO3 mode) and citrate (vCOO mode) ions incorporated into ACP, as previously reported [5], In the case of ACP+BG physical mixtures the FT-IR spectrum is not reproducible due to the non-homogeneities in composition of this material. Also with FT- IR spectroscopy the composite ACP/BG give less clear results than ACP/BGN and ACP/MBGN as 45S5 bioglass contains both phosphate and silicate bands, but nevertheless the findings described above are clearly observable.

Ion release and mineralization of SBF

The key tests to evaluate remineralization capability of ACP/BG composites were (i) their ion release in an acidic artificial saliva that mimics a carious oral environment (modified Tani-Zucchi solution), and (ii) their capability to induce HA crystallization when are in contact with simulated body fluid.

Regarding ion release in artificial saliva, all composites show a release of Ca, P, Si, and F (Figure 3). The release is more intense (burst) for the first 2 hours and remains sustained up to 5 hours, afterward it gradually tapers off continuing as a slow release up to 24 hours. The release curves were compared to pure ACP, which shows only a burst release of Ca, P, and F in less than 2 h, and to pure BG, whose release of Ca, P, and Si is less intense and is spread across the 24 h timeframe.

In the case of ACP/BG composites, their release is intermediate between the ones of pure ACP and BG and depends on ACP:BG ratio, i.e. ACP-rich composites have a more intense Ca, P, and F release and less intense Si release, while in BG-rich composites the behavior is the opposite (Figure 3A-D). This proves that ion release can be modulated by adjusting the ACP:BG ratio. The most important finding is that ACP/BG 60:40 composite has a stronger Ca, P, and F release and a weaker Si release than the ACP+BG physical mixture with identical weight ratio. This is the definitive proof that the composite preparation gives better ion-releasing properties which cannot be achieved by only mixing the two base ingredients.

In the case of ACP/MBGN composites, the observed behavior is the same, but the differences are more attenuated with less distinction between composites, pure MBGN, and ACP+MBGN physical mixture (Figure 3E-G). This is because MBGN, being porous and nanoparticulated, has a stronger ion release than micrometric BG. It is worth noting F’ release of ACP+MBGN physical mixture, which is higher than any composite and is comparable to pure ACP release. This difference proves that composite allows to tune ion release while simple mixing of the base materials cannot.

The other key test to evaluate remineralization capability of ACP/BG composites is the assessment of their capability to induce HA crystallization when are in contact with simulated body fluid (SBF) by the simultaneous (i) crystallization of ACP into HA as well as (ii) the precipitation of calcium and phosphate ions contained in SBF into new mineral by interaction with ACP/BG.

The test was performed on ACP/MBGN composites. By monitoring ion concentration of liquid phase, it was found that all ACP/MBGN composites induce a progressive decrease of Ca, P, and Mg in comparison to initial ion content of SBF in parallel to a release of Si in solution, and with time all curves level off. This means that a precipitation of inorganic minerals occurred.

The amount of precipitated ions is directly proportional to ACP:BG ratio, while Si release is inversely proportional to it. After three days of incubation all phosphate in solution has been consumed, while only a part of Ca and Mg have been removed.

By comparison with pure ACP, it was evident that ACP triggers immediately the precipitation of calcium phosphate due to a seeding effect, as it can be seen it has the highest ion decrease even at short time. On the other hand, the comparison with pure MBGN shows a slower precipitation over time, due to calcium and silicate release from MBGN as first step which later induces the precipitation of calcium phosphate minerals due to exceeded supersaturation.

Therefore, the behavior of ACP/MBGN composites in SBF is controlled by ACP:BG ratio. Observing ion concentration of SBF over time, Ca, Mg, and P data suggest that all composites induce the precipitation of a Mg-doped calcium phosphate phase until all P in solution has been consumed, and the precipitation is faster for ACP and ACP-rich composites.

The comparison with ACP+MBGN physical mixture proves that mixing the two ingredient does not give the same effect, as Ca, P, and Mg precipitation is less intense than with the composite while Si release is more intense. These analyses prove that all composites induce the formation of a new Mg-doped calcium phosphate phase when are in contact with SBF, and ACP component accelerates the mineralization process and enhances Mg and F doping, while the presence of MBGN provides a sustained Si release. In addition, the mineral formation rate can be controlled by adjusting the ACP:MBGN ratio.

The materials after SBF immersion were analyzed by SEM (Figure 4). It can be observed that in ACP/MBGN composites the MBGN particles have not changed morphology, while spherical ACP nanoparticles converted into small, needle-like nanocrystals (Figure 4A-C). These crystals are less than 20 nm wide and less than 100 nm long and are present thorough all the sample. This morphology is similar to pure ACP control sample (Figure 4E), while in pure MBGN was found a formation of thick needle-like crystals onto MBGN particles that with time grow in size as clusters of elongated crystals ca. 200-400 nm long and ca. 50 nm wide (Figure 4D). Is widely reported that BG immersed in SBF induce the formation of HA while ACP converts into HA, therefore the new crystals observed in the composites are HA nanocrystals formed ion release and reprecipitation given by both MBGN and ACP (thicker needle crystals), as well as by ACP direct conversion (thinner needle crystals). In the composites ACP component controls HA formation kinetics, and induces the formation of new HA nanocrystals in proportion to ACP:MBGN weight ratio.

References

1. Cochrane, N.J., et al., New Approaches to Enhanced Remineralization of Tooth Enamel. Journal of Dental Research, 2010. 89(11 ): p. 1187-1197.

2. Neel, E.A.A., et al., Demineralization-remineralization dynamics in teeth and bone. International Journal of Nanomedicine, 2016. 11: p. 4743-4763.

3. Zheng, K., et al., Toward highly dispersed mesoporous bioactive glass nanoparticles with high Cu concentration using Cu/ascorbic acid complex as precursor. Frontiers in chemistry, 2019. 7: p. 497.

4.Kokubo, T. and H. Takadama, How useful is SBF in predicting in vivo bone bioactivity? Biomaterials, 2006. 27(15): p. 2907-2915. 5. lafisco, M., et al., Fluoride-doped amorphous calcium phosphate nanoparticles as a promising biomimetic material for dental remi neralization. Scientific Reports, 2018. 8(1 ): p. 17016.

6. Bari, A., et al., Copper-containing mesoporous bioactive glass nanoparticles as multifunctional agent for bone regeneration. Acta Biomaterialia, 2017. 55: p. 493-504.

7. Combes, C. and C. Rey, Amorphous calcium phosphates: Synthesis, properties and uses 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 spectroscopies. 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 amorphous calcium phosphate particles and bioactive glass particles (ACP/BG), wherein the BG particles are surrounded and immersed in a matrix of ACP particles, thereby forming a homogeneous, intimately and deeply-intertwined composite material.

2. The composite according to claim 1 , wherein the BG particles are macro- or micro-metric particles or nano-particles preferably porous or mesoporous.

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

4. A process to prepare the composite according to anyone of the claims 1 to 3, comprising the following steps: a) Mixing an aqueous solution comprising a calcium salt and/or calcium hydroxide, BG particles and a sodium or potassium citrate salt and/or citric acid (solution A) with a solution comprising a phosphate salt and/or phosphoric acid and a carbonate salt and/or carbonic acid (solution B); b) Stirring the mixture for at least 3 seconds to allow the formation of a precipitate; c) Collecting and drying the precipitate.

5. The process according to claim 4, wherein in step c) the precipitate is dried by freeze-drying.

6. The process according to claim 4 or 5, wherein before step a) solution A is sonicated to disperse the BG particles and the pH of solution B is brought to a basic pH of above 9.

7. The process according to anyone of claims 4 to 6, wherein solution A includes zinc, strontium, iron, silver, copper, sodium, potassium, and/or magnesium ions that are doping agents for ACP; solution B includes fluorine, and/or chlorine ions that are doping agents for ACP.

8. The composite material according to anyone of the claims from 1 to 3, for use in dentistry.

9. The composite material for use according to claim 8, as remineralizing agent.

10. The composite material for use according to claim 9, to prevent dental demineralization and/or to boost dental remineralization.

11 .The composite material for use according to claim 8 to prevent and/or treat dental caries and cavities.

12. The composite material for use according to claim 8 to achieve dentin desensitization by occluding the dentinal tubules with the composite.

13. The composite material according to anyone of the claims from 1 to 3, for use for bone regeneration, preferably as a bone defect filler or in the form of a scaffold or as a coating for orthopedic implants.