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THE ENAMEL REPAIRER

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LEBSC

Laboratorio di Strutturistica
Chimica Ambientale e Biologica

ALMA MATER STUDIORUM
UNIVERSITÀ DI BOLOGNA

Laboratory of Enviromental and Biological Structural Chemestry

I am pleased to present BioRepair® Plus to you.
BioRepair® Plus, developed by Coswell Research Laboratories in collaboration with the LEBSC(Laboratory of Environmental and Biological Structural Chemistry) at the University of Bologna, Italy,
is the first toothpaste based on bioactive microparticles which, thanks to their composition, are able
to penetrate into the microscopic defects on the enamel and dentine surfaces, performing an effective remineralising and reparative treatment.
The LEBSC has been operating for thirty years and uses the most advanced techniques to study thechemical and biological aspects of mineralization processes of calcified tissues and, in particular,bone tissue.
Enclosed is an in-depth scientific study which supports the effectiveness of BioRepair® and its
MICROREPAIR® action
Used daily, BioRepair® will repair tooth enamel and will make your teeth more healthy – definitely
something to smile about!
Thank you for your time.
Yours faithfully,

Prof. Norberto Roveri
Full Professor of General and Inorganic Chemestry“G. Ciamician” Department of Chemistry,
University of Bologna

ALMA MATER STUDIORUM - UNIVERSITÀ DI BOLOGNALABORATORIO DI STRUTTURISTICA CHIMICA AMBIENTALE E BIOLOGICAVIA SELMI, 2 - 40126 BOLOGNA - ITALIA TEL. +39 051-2099486 - 485 - FAX +39 051-2099593 - www.lesbc.unibo.it
Unità Operativa del C.I.R.C.M.S.B. Consorzio Interuniversitario di Ricerca Chimica dei Metalli nei Sistemi Biologici
Via C. Ulpiani, 27 - 70126 Bari (Italia) - P.I. e C.F.: 04434750727 - Reg. Società: 29713 Tribunale di Bari - CCIAA: 314319

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Introduction
Like vertebrate bone tissues teeth are a composite natural material, the main component of which is an inorganic carbonatedhydroxyapatite phase, representing 65-70% of dentine byweight and over 90% of enamel by weight.
Dental enamel forms the thin outer coating of our teeth and isconsidered to be the hardest and strongest of all biogenic materials (figure 1).
Tooth enamel is composed mainly of large, prismatic, highly
crystalline hydroxyapatite microparticles , together with a very
low protein component (figs. 2a and 2b).
Figure 1: Tooth structure

1 µm

5 µm

Figure 2a: Enamel structure viewed with Scanningelectron microscopy (SEM) (Scale bar=5µm).
Reproduced with permission from H. Lowestan and S.
Weiner, "On Biomineralization", Oxford University
Press 1989.

Figure 2b: SEM image: layers of rod-like hydroxyapatite
microparticles that form enamel (Scale bar=1µm).
Reproduced with permission from H. Lowestan and S.
Weiner, "On Biomineralization", Oxford
University Press 1989.

Figure 3: Dentine structure viewed using Scanningelectron microscopy (SEM). The dentinal tubules can beseen clearly, occupied by the odontoblast processes andintratubular fluid implicated.

In adults, enamel does not contain any cells and is therefore not capable of self regeneration. Any damage isirreversible, as there is no biological process capable of repairing damaged enamel. In a similar way, dentine(figure 3) exposed to the oral environment cannot be regenerated, as new dentine is deposited on the interior
surfaces of the crown close to the pulp, and not on the outside. For this reason, any reparative action must be
provided by materials or substances that are extraneous to dental tissue metabolism. These substances areeither synthetic or are precipitated from saliva.

MICROREPAIR®
The hydroxyapatite microparticles (MICROREPAIR®)
present in BioRepair® Plus are completely identical tothe mineral that forms dentine and enamel. It is precisely from this similarity that the synthetic microparticles derive their capacity to reconstruct dentine andenamel.

BioRepair® Microparticles
Ca(10-x)Znx(PO4)(6-y)(CO3)y (OH)2(Y=4-8 % CO32- substituted at PO43-)(X=1 % Zn2+ substituted at Ca2+)
Figure 4: Chemical formula for dentine and enamel remineralising hydroxyapatite
microparticles (Microrepair®). Calcium phosphate and carbonate are present in very
similar relationships to those occurring naturally. It is the zinc, with its antiseptic
properties, that is responsible for the plaque-preventing action.

The substance used in BioRepair® Plus is technologically innovative, in that being in the form of microparticles
it has enhanced chemical reactivity. Microparticles carry out their dentine remineralising action by releasing
their constituent calcium and phosphorous in situ. In the case of the enamel, the microparticles action takes
place via their ability to bond to natural tissues, thus filling microgaps in the enamel.

Figures 5 and 6: Microscopic Microrepair® microparticles aggregates viewed using TEM(Transmission electron microscopy) (Scale bar=100 µm)

Figures 7 and 8: Microscopic Microrepair® microparticles aggregates viewed using TEM (Transmission
electron microscopy (Scale bar=50 and 20 µm respectively).

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Introduction
Action mechanism
The enhanced reactivity of MICROREPAIR® microparticles is due primarily to the bio-mimetic
action of these microparticles, which have thedistinguishing feature of possessing a very similar
chemical composition to that of enamel anddentine. The X-ray diffraction spectrum of
MICROREPAIR® microparticles (figure 9) shows
how the degree of crystallinity of the microparticles is half way between that of enamel anddentine.
Figure 9: X-ray diffraction diagrams of enamel (red line),
MICROREPAIR® microparticles (blue line) and dentine (greenline).

Figure 9

The ability to remineralise hard tissues has an impact on several disorders that affect the hard tissue of teeth:
1. Prevention of cavities by remineralisation of original lesions;
2. Desensitising effect on dentine by covering up dentinal tubules;
3. Tartar and plaque prevention thanks to antibacterial action of Zn2+, with recognised antiseptic properties
Figure 10: the SEM images show the surface of thedentine before treatment (a) and after treatment
with MICROREPAIR®: 1 minute (b), 10 minutes (c)and one hour (d). They clearly show the progressive
growth of the apatite microparticles until they
completely hide the dentine channels.

Figura 10aFigura 10b

Figure 10a

Figure 10b

Figura 10c

Figura 10d

Figure 10c

Figure 10d

Figures 10 (a, b, c and d) show the progressive action of MICROREPAIR® microparticles, as they gradually bondsecurely to the surface of the dentine, blocking the tubules and thus performing their effective, long-lasting
desensitising effect on the dentine.
The phenomenon can be seen at work a few minutes after the BioRepair® Plus microparticles have been applied,which means that the microparticles remineralising and desensitising action begins after only a few applicationsof BioRepair® Plus toothpaste.
A similar mechanism can be seen on the surface of the enamel where there are microscopic defects or simple
surface irregularities, as the BioRepair® Plus microparticles are laid down and begin their recrystallisation
process.
The Zinc release mechanism is extremely innovative, as it takes place via slow dissolution of the apatite, whichthen releases its various components locally (Zn2+, Ca2+, CO32-, PO43-). The Zinc, in its ionic form as a bivalentaction, then performs its antiseptic function inside the mouth.
The Zinc is released at an optimum concentration, therefore carrying out its antiseptic action in the mouth.
4. Finally, apatite has other secondary effects, including the absorption of sulphate compounds such as H2S,
which is responsible for halitosis. Daily use of BioRepair® Plus is therefore also indicated for control of this condition: thanks to the absorbent properties of BioRepair® Plus microparticles , daily use of Microrepair® is effective
in fighting halitosis problems.

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Repairing Enamel
N. Roveri, E. Foresti, M. Lelli, I.G. Lesci
Laboratory of Environmental and Biological Structural Chemistry
Department of Chemistry “G. Ciamician”, University of Bologna,
via Selmi 2, I-40126 Bologna, Italy
norberto.roveri@unibo.it www.lebsc.unibo.it
Teeth
Enamel is the hardest material in vertebrates and is the most highly mineralized skeletal tissue present in the
body [1]. Mature enamel is composed of carbonated hydroxyapatite (CHA) (95-97%wt) and less than 1% wt
of organic material. Unlike other biomineralized tissues, such as bone and dentine, mature enamel has nocells and therefore cannot be biologically remodelled. Consequently, enamel regeneration cannot take placein vivo. Affording this situation is therefore an attractive target for future innovative biomimetic and therapeutic approaches for the health protecting possibilities involved.
The mammalian tooth is made up of four
distinct structures: enamel, dentine, pulp,
and cementum.
Enamel (E) is the outermost layer on thecrown of the tooth and situated above thedentine (D). The pulp (P) contains nerves
and blood vessels, while the cementum (C)is the outermost layer of the mineralized
tissue surrounding the root of the tooth
allowing the tooth, keeping it anchored tothe jawbone through the periodontal ligament (Fig. (1)) [2].
The pulp contains nerves, blood vessels,
fibroblasts and lymphocytes, while thetooth mineralized area concerns enamel,
dentine and cementum. Enamel makes up
the uppermost 1-2 mm of the tooth crownand contains a high mineral content, givFig.1 Tooth anatomic draft: enamel (E), dentine (D), pulp (P) and cementum (C).

ing it a high modulus, but also making it
susceptible to cracking. Dentine lies below

the enamel and is tougher, forming the bulk of the tooth and absorbing stresses from enamel, preventing
its fracture [3]. The cementum is the mineralized layer that surrounds the root of the tooth covering the dentine layer and anchoring the tooth to the alveolar bone (jawbone) through the periodontal ligament. The
primary function of the teeth is for mastication of food and it is very important to maintain them healthy
keeping their mechanical properties long life lasting in a bacteria-filled environment.
Enamel prismatic HA microparticles consist of a weaving of prisms ranging from 3 to 5 μm in diameter. A single
prism reveals a highly organized array of fastened needle like HA crystallites (approximately 30 nm thick, 60
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nm wide, and several millimetres long) (Fig. (2A)). They are preferentially aligned along the HA crystallographic c-axis organized in interweaving bundles of aligned crystallites that are “woven” into intricate architectures approximately 3-5 μm in diameter (Fig. (2B)) [4].

Fig.2 Scanning Electro Microscopy enamel images of highly organized array of fastened needle like HA crystallites (A) organized in interweaving bundles into intricate architectures (B).

The composition and morphology of dentine resembles that of bone and is characterized by exhibition ofnumerous tubules containing nervous tails (Fig. (3)).
Enamel and dentine are tough, crack-tolerant, and abrasion-resistant tissues fortheir unique architectures and mineralcompositions. Because of the high mineralcontent and minimal organic one, enamelis brittle. It is interesting to note that thearchitecture of the enamel crystallites candeflect a propagating crack, preventing
this from reaching the dentine-enamel
junction (DEJ), a gradual transition fromdentine to enamel and has been shown toresist to the tissues de-lamination despite
their different composition [5].
Fig.3 Scanning Electro Microscopy image dentine showing
tubules in a bone like morphology.

Teeth hearth prevention
Dental erosion is the chemical wear of the dental hard tissue without the involvement of bacteria [6]. Its clinical relevance is becoming wider and wider [7-11], and it is considered one of the main tooth pathologies able
to cause patient discomfort, after periodontal diseases and caries.
Erosion aetiology appears related to the enormous increase in consumption of soft drinks, fruit juices andsport drinks consumption [12]. However, other acid sources, such as drugs containing syrups, analgesics andvitamin C intake, and environmental acid exposure in working condition are claimed to be related to enamelerosion development [13-17].
The mechanisms involved in the damage of dental hard tissue are related to the acid attacks on the outer few
micrometers of the enamel, with the consequent demineralization and dissolution of the minerals [18-24].
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Dentine hypersensitivity, defined from Holland et al. [25] as “the short sharp pain arising from exposed dentine in response to stimuli typically thermal, evaporative, tactile, osmotic or chemical and which cannot beascribed to any other form of defect, pathology or disease” [26], has been explained in term of hydrodynamic mechanism [27]. Essentially, if dentine is exposed and the dentine tubules are open, the fluid movement provokes stimulation [28]. Even if much remains unknown or unproven about the aetiology of dentine
hypersensitivity, according to many authors it can be considered a tooth wear phenomenon [29]. The smear
layer, which is an artificial surface about one micron thick, consisting of collagen and hydroxyapatite fromthe native dentine, is formed when dentine is abraded [30]. It covers the underlying dentine occluding thetubules, but can be removed by attrition, acid erosion and tooth brushing with toothpaste [31,32]. Therefore
a considerable overlap exists between the aetiology of dental erosion and that of dentinal hypersensitivity.
To prevent dental erosion progression, the reduction or elimination of exposure to acidic soft drinks andjuices can be recommended to patients. Frequent application of a high concentration of topical fluoride may
be of some benefit in preventing further demineralization and increasing the abrasion resistance of erosion
lesions [33]. In–vitro studies have shown that inhibition of dissolution of synthetic carbonated-hydroxyapatite is a logarithmic function of the fluoride concentration in solution [34].
The demineralised area and micrometric sized scratches normally occur on enamel surface as a consequenceof micro wear and acid attack [35] and cannot be repaired biologically, nor prosthetically. In fact, unlike inbone, when apatitic microparticles are dissolved or abraded, they cannot be spontaneously re-deposited inenamel and dentine because enamel contains no cells able to secrete extra-cellular matrix. The regenerationand dentine apposition occurs only through the pulp tissue. Therefore, both enamel and dentine can bereconstructed only by the application of all plastic materials which provide a sort of prosthetic restoration.
Most of the products and devices commonly used to counter enamel and dentine erosion such as fluoride[36-39], work by reducing apatite dissolution and increasing surface micro hardness [40- 42], but are unable
to reconstruct the lost mineral.
Hydroxyapatite is the main constituent of the dental tissues representing in enamel and dentine the 95 wt% and 75 wt % respectively and, like in bone, it is the main responsible for the mechanical behaviour of thedental tissues.
Hydroxyapatite has been widely experimented as a biomaterial, thanks to its biocompatibility and osteoconductivity. It represents an elective material covering a wide range of biomedical applications for bone substitution and bone-prosthesis interface [43]. Poorly crystalline HA microparticles, in addition to the excellent
biological properties of HA (non-toxicity and lack of inflammatory and immunizing responses) can bebiodegradable in physiological conditions. This property can be modulated by modifying its degree of crystallinity, which recently can be achieved by implementing innovative synthesis able to control crystal growthat nano size level. In the last decade, advanced technology has been utilized to synthesize a new generationof biomimetic apatitic all plastic materials which can optimize the interaction with biological entities thanks
to their strong surface bioactivity [44]. The aim of this paper is to review the main patents concerning oral
care fluoridated products and the recent patents in which a daily use of synthetic apatite instead of fluoride
for health care is recommended.
Remineralization by fluoride
In vitro, fluoride (0.02-0.10 mg/L) addition to a supersaturated solution of calcium phosphate induces the
crystallization of hydroxyapatite, Ca10(PO4)6(OH)2 which is the mineral phase of bone and teeth. Increasing
fluoride concentration fluoroapatite Ca10(PO4)6F2 is formed and appears in more ordered and bigger
apatite microparticles which are less acid soluble [45-47].
In the body, in vivo fluoride is mainly associated with calcified tissue, bone and teeth due to its high affinity
to calcium. Fluoride modifies the bone mineral phase by replacing the hydroxyl groups in hydroxyapatite
phase producing its partial conversion into fluoroapatite. The more electrostatic stability and crystallinity offluoride substituted hydroxyapatite increases the bone density and hardness reducing the mechanical8BrochueProva:Layout 1

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strength [48,49]. Fluoride in high doses was found to be mutagenic in osteoblasts and inhibitory of osteoclasts [50-52,48].
In spite of the observed negative physicochemical effects on the bone, fluoride exhibits both a cariostatic
effect on children and adults erupted teeth and a pre-eruptive effect through increasing fluoridation of thedeveloping enamel [53,54]. Fluoridated enamel is less acid soluble [55]. The reduction of caries in both deciduous and permanent teeth was more marked where the children were earlier exposed to fluoridated tip
water [53,56]. The fluoride cariostatic effect on erupted teeth can be ascribed to an inhibition of the demineralization of sound enamel due to ingested acid foods and drinks and cariogenic bacteria in the dental
plaque. Sound enamel results more acid resistant containing more fluoride [57,58]. However, caries is not afluoride deficiency disease and no specific fluoride deficiency syndrome has been found.
Adsorbed fluoride is rapidly distributed by circulation to the intracellular and extracellular fluid, is retained
only in calcified tissues and the fluoride plasma concentration is dependent on the ingested fluoride dose.
Fluoride concentrations in oral environment and glandular saliva closely follow the plasma concentration,
but at a lower level about two-thirds of the plasma level [59]. This finding suggests that the fluoride concentration in saliva and dental plaque is strongly dependent on topical fluoride application by dental care products [57,60-74]. Children with no caries experience were found to have higher salivary fluoride concentrations than children highly affected by caries [75].
Contrary to skeletal bone and dentine which accumulate fluoride in proportion to the fluoride adsorbed during life, enamel reflects the biologically available fluoride during teeth formation.
Enamel maturation of deciduous teeth is completed between the age of 2 to 12 months while in permanent
teeth enamel maturation is completed at the age of 7-8 years except for the third molars whose maturation
continues until the age of 12-16 years. Post-eruptive fluoride uptake of enamel is expressed only in the outerlayer and depends on fluoride in saliva, food, drinks, dental plaque and prevalently in dental products. In
order to prevent enamel demineralization by acid food and drinks and cariogenic bacteria in the dental
plaque, many dental products as toothpaste, rinses and gels containing fluoride have been recommended by
medical societies in many countries for caries prevention. The use of fluoridated oral care products are recommended especially in countries where the fluoride concentration from drinking water is low even if there
are considerable differences regarding the starting time (birth-6 months of age) and amounts related to age.
However fluoride from toothpaste swallowed by a four year old child was found to contribute up to one
third to one half of total daily fluoride intakes of 3.6 and 2.3 mg respectively [76]. In the European communities about 90% of all toothpastes are fluoridated in the range from 1000 mg/kg to 1500 mg/kg with a maximum level of 1500 mg/kg. The Scientific committee on Cosmetic products and non-Food Products Intended
for Consumers (SCCNFP, 2003) states that the amount of toothpaste applied to the toothbrush for a childbelow the age of 6 years can vary between 0.05 and 0.8 g. The recommended “pea size” amount is considered to be 0.25 g. According SCCNFP simulation model the fluoride ingestion from such toothpastes could
amount up to 50% of the adequate fluoride intake of children at that age. In the model calculation for 3-5
year old children in the USA the fluoride intake from ingested toothpaste was estimated to be 30-60% of
the dietary [77].
Fluoride tolerable intake
Fluoride is not essential for human growth and development and its content in the body is not under physiological control. Adsorbed fluoride is rapidly distribute by circulation to the intracellular and extra cellular
fluid, but is retained only in calcified tissues. Plasma fluoride occurs in both ionic and non-ionic forms which
consists mostly of fat-soluble fluorine compounds. In adults, adsorbed fluoride is only partially less than 50%
retained in skeleton and the remainder excreted prevalently via the kidney. On the contrary, in infants fluoride retention in bone can be as high as 90% and appears also incorporated into dental enamel during teeth
formation. Excessive intake of fluoride during enamel maturation from birth to eight years of age , whenenamel formation is complete, can lead to reduced mineral phase content of enamel and to dental fluoro9

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sis of deciduous, but prevalently of permanent teeth. Dental fluorosis has been usually associated withincreased resistance to caries, but it is increasing the consideration of appreciable fluorosis as an adverse
effect [78]. While mild dental fluorosis is not readily apparent, moderate dental fluorosis can be easily appreciated and is characterized by white spots and opaque striations staining and minute pitting of teeth (Fig.
(4)) [79]. Fluorides in toothpaste, whilst well known for their anti-caries benefits, are toxic if ingested at highlevels, in particular in children because of an adverse dose to weight ratio [80,81].
The International Standards Organization Toothpaste minimizes this safety matter by fixing the maximum
fluoride dose allowed in oral care products. Furthermore, children products contain a lower fluoride concentration, and therefore a lower dose, and ⁄ or manufacturers recommend a small amount of adult toothpaste
to be placed on the brush. The recently produced toothpastes containing a very high fluoride concentration
exceeding the International Standards Organization limits are exclusively prescribed by professionals and are
not recommended for children [82]. The European Food Safety Authority (EFSA) Scientific Panel considers
that the maximum fluoride intake is 0.1 mg fluoride/kg/day in children aged 1-8 years which is equivalent to
1.5 and 2.5 mg fluoride per day in children aged 1-3 years and 4-8 year respectively.
Fluoride accumulation in skeleton changes bone mechanical behaviour reducing bone strength and increasing its density and stiffness, causing skeletal deformities and risk of fractures. Therapeutic studies with fluoride in postmenopausal osteoporosis suggest an increasing risk for skeletal fractures at or above fluoride
intakes of 0.6 mg/kg body weight per day. Fluoride increases with age in bone, more rapidly in women thanin men and preferably in cancellous bone [83,84].
Fluoride is not irreversibly bound to bone and can be released during remodelling of bone [85].
Excluding the fluoride exposure via inhalation and the skin which in normal circumstances is really negligible, fluoride intake is due to oral ingestion by drinking water, beverages, foodstuffs, including fluoridated
salt, dental health products and fluoride tablets for caries prevention.
Fluoride concentration in drinking water (0.3-1.5 mg/L) differs according to the countries’ natural circumstances and to water fluoridation (U.K., Ireland, Spain and Switzerland). This has been recently reduced or
terminated. In a 2004 Water Quality Report for a certain public water supply, the following Public Notice wasissued regarding Fluoride: "This is an alert about your drinking water and a cosmetic dental problem thatmight affect children under nine years of age. At low levels, fluoride can help prevent cavities, but children
drinking water containing more than 2 mg/L of fluoride may develop cosmetic discolouration of their teeth(dental fluorosis). Dental fluorosis, in its moderate or severe forms, may result in a brown staining and/or pit-

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ting of the permanent teeth (Fig.(4)).

Fig.4 Fotografic images of teeth affected by dental fluorosis.

This problem occurs only in developing teeth, before they erupt from the gums. Children under nine should
be provided with alternative sources of drinking water or water that has been treated to remove fluoride to
avoid the possibility of staining and pitting of their permanent teeth. You may also want to contact yourdentist about proper use by young children of fluoride-containing products. Older children and adults may
safely drink the water. Drinking water containing more than 4 mg/L of fluoride (the U.S. Environmental
Protection Agency’s drinking water standard) can increase your risk of developing bone disease. Your drinking water does not contain more than 4 mg/L of fluoride, but we’re required to notify you when we discover that the fluoride levels in your water exceed 2 mg/L because of this cosmetic dental problem. Some homewater treatment units are also available to remove fluoride from your drinking water" [79].
Vegetables and fruit containing from 0.02 to 0.20 mg/kg fresh weight, milk and milk products 0.05-0.15
mg/kg, meat and meat products 0.15-0.29 mg/kg, eggs 0.18 mg/kg, fish 0.48-1.91 mg/kg.
Exceptions are tea which can contain considerable amounts of fluoride (0.34-5.2 mg/L), and some brands of
instant teas were reported to contain significant amounts of fluoride even 6.5 mg/L .On the contrary, inhuman milk the fluoride concentration is about 0.2 mg/L.
Children aged 1-8 years get fluoride intakes from food and water usually far below the UL. The clear increaseof mild dental fluorosis occurred in some countries has been ascribed to the inappropriate use of fluoridated dental care products in particular fluoridated toothpastes. Dental products like toothpaste, rinses and gels
containing fluoride can increase the total intake of fluoride especially when inappropriately used [86]. This
happens especially in children younger than 7 years ingesting high amounts of toothpaste [87-90]. In themodel calculation for 3-5 year old children in the USA the fluoride intake from ingested toothpaste is estimated to be 30-60% of the dietary CTE.
For these reasons, in the European Communities all toothpastes are fluoridated with a maximum level of1500 mg/kg. The need to discover and develop an alternative to fluoride for teeth health prevention care isclearly opportune.

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Biogenic hydroxyapatite
Vertebrate bones and teeth are biological hybrid materials where a calcium phosphate, in the form ofhydroxyapatite (HA), represents the inorganic component intimately inter grown with the organic matter
prevalently constituted of proteins and polysaccharides [91,92]. Biological HA is not stoichiometric according
to the ideal formula Ca10(PO4)6(OH)2, but at low extent Ca2+ is replaced by other ions like Zn2+, Sr2+, Na+, K+,
Mg2+ while PO4 3- and OH- can be partially substituted by other anions like CO3 2-, HPO4 2-, P2O7 4-, SiO4-.
The bone mineral phase is more correctly called carbonate hydroxyapatite. Carbonate is the prevalent foreign anion and represents about 4-8 wt % [93,94]. The substitution of CO3 2- groups into the PO4 3- sites (type
B carbonate apatite) is prevalent in young humans, while the carbonate replacement to OH- groups (type A
carbonate apatite) increases with the age of the individual [95].
The bone carbonate hydroxyapatite microparticles, which can represent a typical example of an “organic
matrix-mediated” biogenic material, have a blade shape of approximately 25 nm width, 2 - 5 nm thickness
and about 60 nm length. Biogenic hydroxyapatite microparticles exhibit nonstoichiometric composition,
structured carbonate ions in the crystal lattice, low degree of crystallinity, plate acicular morphology and anano size which give a large surface area of about 120 m2/g (Fig. (5)) [92,96,97].
Vertebrate bone can be considered a “living biomaterial” since it contains inside a network of different cells
under permanent activity, living within the mineralized structure, and are interconnected through pores andchannels. A dynamic process of bone formation and destruction accounts for its growth during the bodydevelopment and regeneration after fractures.
Dentine, resides within the central region of the tooth and is similar to bone in composition and structure[98-107]. Enamel, the tooth external surface coating, has a much larger inorganic content than bone andFig.5 Transmission Electron Microscopy image of deproteinated bone hydroxyapatite microparticles.

dentine, close to 95% wt, which is mainly constituted of long thin ribbon-like prismatic microparticles ofhydroxyapatite, that exhibit a higher degree of crystallinity and a lower carbonate content than bone anddentine apatite microparticles. Amelogenins, present in relatively large amount in the early stages of enamel formation, are enzymatically degraded and removed up to 5% wt as the hydroxyapatite microparticles
grow [108]. Adult dental enamel, considered the most resistant and tough material in the biological world,
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does not contain cells and therefore cannot be regenerate itself. There is no biological process that canrepair degraded or damaged enamel, evidencing the need for synthetic enamel biocompatible materials
able to repair teeth decay [109-111].
Biomimetic synthetic hydroxyapatite
Biomimetism of synthetic materials for biomedical applications can be carried out at different levels in viewof composition, structure, morphology, bulk and surface chemical-physical properties.
Biomaterials can be turned biomimetic imprinting all these characteristics in order not only to optimize theirinteraction with biological tissues, but even to mimic biogenic materials in their functionalities. Chemists,biologists, physicists and engineers interested in material science are amazed by the high degree of sophistication, miniaturization, hierarchical organization, hybridising, reliability, efficiency, resistance and adaptability characterizing natural materials.
These properties which biogenic materials have achieved through specific building principles selected by evolution, can be only partially obtained in manmade materials by present synthetic processes. For this reason
Nature is a school for material science. Biomimetism and bioinspiration represent important tools for thedesign and the synthesis of innovative materials and devices [112- 116].
The highly elaborated performances of biologically occurring materials are the results of an evoluted convergence on limited constituents, which occur at a precise moment, and are available at that time. Nature
produces soft and hard materials exhibiting remarkable functional properties by controlling the hierarchicalassembly of simple molecular building blocks from the nano to the macroscale [117]. Biomineral morphogenesis is related to specific strategies for the long-range chemical construction of well organized architecturesfrom preformed nano or micro crystalline inorganic building blocks. In fact, many biologic complex structures
are obtained by promoting specific links induced by the conformation variability at the nanometre scale ofbiological macromolecules. Biosystems reveal a high level of integration of three fundamental aspects: thenano-micro “spatial confinement” of biochemical reactions, the inorganic and organic “hybridization” compounds and the “hierarchy” from nano to macro scale, in order to produce a biomaterial able to exhibit theappropriate chemical-physical properties at any different scale level [118-121]. Biogenic materials are nucleated in defined nano-micro dimensioned sites inside the biological environments in which chemistry can bespatially controlled. The spatial delimitation is essential to biological mechanisms to control size, shape andstructural organization of biomaterials.
With the development of nanotechnology, this strategy employing natural material genesis, has attracted a
lot of attention in designing bioinspired materials such as polymeric micelles, microparticles, dendrimers andmicroparticles synthesized in nanoscale dimensions [122-127].
Since the last 30 years calcium phosphate ceramics have been, and still are nowadays, very popular implantmaterials for diverse clinical applications. Porous HA, simulating spongy bone morphology has been prepared using various technologies to control pore dimension, shape, distribution and interconnections. HA
ceramics processed by high-temperature treatment [128] present a significant reduction of bioreactivity andgrowth kinetics of new bone due to the not resorbability. New synthetic methods at lower temperatures
have been developed, allowing one to obtain porous bioceramics with a low degree of crystallinity. Colloidal
processing [129], starch consolidation [130], gel casting and foam out [131] have yielded excellent results,
producing bioceramics with a bimodal distribution of the pore size that can be modified as a function of thesintering conditions.
Different types of CaP-ceramics are available, though they can be classified as either hydroxyapatite (HA),
beta-tricalcium phosphate (β-TCP), biphasic calcium phosphate (BCP), amorphous calcium phosphate (ACP),
carbonated apatite (CA) or calcium deficient HA (CDHA) [132]. The use of these materials for tissue engineering purposes is still explored. Most researchers are aware that the low resorbability of sintered CaP-ceramics, and in particular the incomplete resorbability of ceramic HA, appears useful when biomaterial has to be
implanted with a defined 3D form. The use of highly porous implants induces bone formation inside the13BrochueProva:Layout 1

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implant and increases degradation, but the complete resorption in most cases is very difficult due to the crystalline architecture.
Porous coralline HA can be synthesized by a hydrothermal method for HA formation directly from naturalsea corals [133] and HA replaces aragonite whilst preserving its porous structure. The coral can be fully converted into hydroxyapatite and then coated with a sol-gel-derived apatite. This new material can be applied
to bone graft applications where high strength requirements and longevity are pertinent [134,135].
When powder bioceramics are used for bone filling applications, they are usually mixed with a polymeric carrier matrix to avoid migration out of the implant region [136].
Both non-absorbable (polymethyl methacrylate [137], polyethylene [138] and polysulfone) and biodegradable (polylactic acid [139], polyglycolic acid, collagen, cellulose and starch [140-142]), polymeric matrices canbe used, even if the non-biodegradability drastically reduces the HA crystal bioactivity.
Synthetic biodegradable biomimetic hydroxyapatite nano and micro microparticles exhibit excellent properties as bone filler biomaterial, such as biocompatibility, bioactivity, osteoconductivity, direct bonding tobone, etc., exciting new applications of HA in the fields of bone tissue engineering and orthopaedic therapies [143,144]. There are many synthetic strategies to produce HA microparticles, including wet producing,hydrothermal, electrochemical and ultrasonic mobilization methods, solgel and solid state synthesis. HA
microparticles with different stoichiometry and morphology have been prepared and the effects of varying
synthesis conditions on stoichiometry, crystallinity, morphology, surface properties, reactivity and bioactivity
has been investigated [145-149] In order to optimize its specific biomedical applications, especially new boneformation and drug delivery function, the physical-chemical features which should be tailored in synthetic
biomimetic HA microparticles are dimensions, porosity, morphology and surface properties [150-153].
The chemical and biological properties of HA microparticles are strictly linked to their dimensions, the regulation of which requires a high level of biological and chemical control at the nano scale. Thus, the recent
trend in biomaterials research is focused on overcoming the limitations of calcium phosphates, preciselyhydroxyapatite ceramics, and in improving their biological properties via exploring the unique advantagesof nanotechnology [154]. The trend is shifting towards nanotechnology to improve the biological responses
of HA, because nano-HA is a constituent of bone improving the biomaterial-bone interface. It has been
established that biomimetism offers a unique approach to overcome many traditional materials shortcomings. Nanostructured biomimetic materials offer much higher performances than their larger particle sizedcounterparts, due to their large surface to volume ratio and unusual chemical/electronic synergistic effects.
In addition, the surface adsorption properties of these materials led to applications in affinity chromatography [155], wastewater remediation [156] and drug delivery systems [149,157,158]. The surface functionalization of HA microparticles with bioactive molecules makes them able to transfer information to and to act
selectively on the biological environment, and this represents a main challenge for innovative bone substitute materials. In this way HA microparticles will not only guarantee, for instance, either osteointegration or
osteoinduction enhanced properties but they will also perform at the molecular level, by stimulating specific cellular responses.
Only in recent years scientists have begun to use biomolecules for the synergistic coupling of microparticles
synthesis and functionalization. In fact, previous studies have limited the use of biomolecules as simple
growth inhibitors of HA crystallization, rather than considering their use as a strategy to fine-tune the bioactivity of the microparticles [159,160]. Studies of the effect of biological molecules onto hydroxyapatite crystal growth have been related directly to physiological or pathological calcification processes. The exposureof biomaterials to plasma proteins, blood or biological fluids normally leads to the adsorption of blood proteins onto the biomaterial surface.
The adsorbed protein layer can further mediate additional biological responses, such as cell attachment andactivation, and can create unpredicted perturbations to device operation [161-168].

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Synthesis and characterisation of biomimetic carbonate-hydroxyapatite microparticles
Biomimetic carbonate-hydroxyapatite microparticles (CHA) have been synthesized with a nearly stoichiometric in bulk Ca/P molar ratio of about 1.6-1.7 and containing 4±1 wt% of carbonate ions replacing prevalently phosphate groups. CHA microparticles have been synthesized both about 100nm and 20 nm sized with anacicular and plate morphology respectively. TEM images of synthetic CHA microparticles 20 nm sized showing the plate shaped morphology and synthetic CHA microparticles 100 nm sized showing the acicular morphology are reported in Fig 6(A),(B) respectively.
CHA microparticles can aggregate in micro sized crystal clusters, whose dimensions increase prolonging maturation time in mother solution at constant temperature and stirring [169]. Powder X-ray diffraction patternsof plate shaped about 20 nm sized CHA microparticles and acicular shaped about 100 nm sized CHA
microparticles (Fig. (7)(b,c)) respectively) show characteristic diffraction maxima of an apatite single phase(JCPDS 9-432). These X-ray diffraction patterns are compared withthose collected for natural carbonate hydroxyapatite from deproteined dentine and enamel reported in Fig.(7)(a,d) respectively. The
broadening of the diffraction
maxima present in the X-ray diffraction patterns reported in Fig.
(7)(a,b,c) indicate a relatively low
degree of crystallinity, The degreeof crystallinity of synthesizedabout 20 nm sized CHA microparticles with plate morphology andsynthesized about 100 nm sized
CHA microparticles with acicularmorphology is 30% and 50%
respectively.
The crystallinity degree of about
20 nm sized CHA microparticles isvery close to that one determinedfrom the X-ray diffraction patternof deproteined dentine naturalcarbonatehydroxyapatite (28%).
Fig.6 TEM images of synthetic CHA microparticles 20 nm sized showing the plate-shaped
morphology (A), synthetic CHA microparticles 100 nm sized showing the plate-acicularmorphology (B), (scale bar =200nm).

Furthermore

thecrystallinity

degree of natural hydroxyapatiteof deproteined enamel is 70%. X-

ray diffraction investigation reveal that the crystal structures of the synthesized CHA microparticles are very
close to those observed for natural dentine.
The same similarity can be observed from the comparison of the FTIR spectra of synthesized CHA microparticles and natural apatite of deproteined dentine reported in Figure 6a and b respectively. In these spectra
the characteristic absorption bands of phosphate and carbonate groups are clearly resolved. The absorption
band at 1468 cm-1 is related to the carbonate group substitution to the phosphate one, while the shoulder
at 1545 cm-1 can be considered the contribution of the carbonate group substituting the hydroxyl group in
the apatite structure. This finding reveals that synthesized CHA microparticles not only contain a similar carbonate amount, but also, underline that the carbonate substitution to the phosphate and/or hydroxyl group15

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Fig.7 X-ray diffraction pattern of natural carbonate-hydroxyapatite from deproteinated dentine (a), synthetic CHA
about 20 nm sized plate shaped microparticles (b), synthetic CHA about 100 nm sized plate-acicular shaped microparticles (c), and natural carbonate-hydroxyapatite from enamel (d).

is very similar in the synthetic and biological microparticles revealing that it can be considered a type B carbonate apatite.
A surface characterization of the synthetic carbonate-hydroxyapatite microparticles has been carried out inorder highlight their surface chemical-physical characteristic which directly interfaces and reacts with
exposed dental tissues. The ATR spectra of the synthetic about 20 nm and 100 nm sized CHA microparticles,
reveal a 4% and 3% wt surface carbonate respectively. The consistent amount of surface % wt of carbonate
present in synthetic CHA is appreciably higher than the surface % wt of carbonate present in enamel anddentine about 2% wt.
Specific surface area of 100 m2g-1 and 80 m2g-1 has been determined for 20 nm sized CHA microparticles withplate morphology and synthesized 100 nm sized CHA microparticles with acicular morphology respectively.
These specific surface area values obtained for synthetic microparticles are only slightly lower than the 110
m2g-1 obtained for biological microparticles.
The surface Ca/P molar ratio determined by XPS analysis for CHA microparticles and CHA microparticles
micro-clusters do not reveal appreciable differences and result significantly lower than Ca/P molar ratio
determined by ICP analysis in bulk indicating a surface calcium deficiency probably due to surface disorder.
In fact the Ca/P molar ratios of 1.7 determined in bulk for synthetic CHA microparticles reduces to a value of
1.4-1.5 when determined on the microparticles surface by XPS analysis [170].
Teeth remineralization by biomimetic carbonate hydroxyapatite
The dentine remineralizing effect of sintetic biomimetic CHA nanometric crystal has been studied with ascanning electron microscopy putting a CHA nano microparticles slurry solution onto slices of dentine previously demineralized with ortophosphoric acid. The HA application shows the progressive closure of the tubular openings of the dentine with plugs within 10 minutes and a regeneration of a surface mineral layer within 6 hours (Fig. (8A-D)).
Biomimetic hydroxyapatite microparticles were demonstrated able to remineralize the surfaces of the dentine etched by orthophosphoric acid application and able to progressively occlude dentine tubules in few
minutes till a regeneration of a layer of mineralized tissue within few hours. This remineralization rate seems
to be compatible with the development of toothpastes with remineralizing effect and able to contrast dentine hypersensibility [171].
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Fig.8
Scanning
ElectroMicroscopy images showing
the features of the dentine
surface demineralised (A),to remineralized specimens
after application of HA
nanocristals slurry solution
for 10 min (B), 1 h (C) and 6
h (D). The pictures show a
progressive microparticles
formation and consequent
obliteration of dentinal
patent tubules.

Scanning Electron Microscopic analysis allows investigating the morphology of both demineralized enamel
and the features observed after remineralization procedures induced by biomimetic HA microparticles invitro application. In fact after treatment for 10 minutes by aqueous slurry of synthetic 100 nm sized CHA
microparticles, the surface of the demineralized enamel by ortophosphoric acid 37% for 1 minute, appears
partially covered by the CHA phase and the interprismatic and prismatic enamel structure become not completely hidden. In contrast, the surface treated for 10 minutes by aqueous slurry of synthetic 20 nm sized CHA
microparticles, the interprismatic and prismatic enamel structures appear to be covered by a thicker andmore homogeneous apatitic layer [172]. This finding reveals an advantage of the 20 nm sized synthetic building block in respect to the 100 nm sized in producing an apatitic coating on the enamel surface.
XPS analysis of spectral features of the O 1s region of the enamel demineralized by ortophosphoric acid 37%
for 1 minute compared with that of the enamel remineralized by synthetic 20 and 100 nm sized CHA
microparticles for 10 minutes unequivocally confirm the presence of synthetic CHA at the surface of thetreated enamel and the consequent validation of the enamel remineralization. The same finding is pointed
out by the ATR spectrum of enamel treated for 10 minutes by synthetic 20 and 100 nm sized CHA microparticles, showing appreciable higher intensity of the characteristic absorption bands of carbonate ions (at 14201460 and 1680 cm-1) in respect of the same absorption bands present in the demineralized enamel ATR spectrum, revealing that the surface of remineralized enamel is richer in carbonate than the natural one, like synthetic 20 and 100 nm sized CHA microparticles [172].
It has recently been revealed that the basic enamel building blocks are 20–40 nm. HAP microparticles and it
has been suggested that the enamel repairing effect of HAP can be greatly improved if their dimensions canbe reduced to the scale of the natural building blocks. Compared with conventional HAP and nano amorphous calcium phosphate (ACP), in vitro experimental results demonstrate the advantages of 20 nm HAP inenamel repairs. Scanning electron microscopy, confocal laser scanning microscopy, quantitative measurementof the adsorption, dissolution kinetics and nanoindentation show the strong affinity, excellent biocompatibility, mechanical improvement and the enhancement of erosion-free by using 20 nm particles as the repairing agent.
However, these excellent in vitro repairing effects cannot be observed when conventional HAP and ACP areapplied. Clearly, 20 nm nano HAP share similar characteristics of the natural enamel building blocks, so thatthey may be used as an effective repairing material and anticaries agent.
Our current study highlights the analogies of nano building blocks of biominerals during biomedical applications, which provide a novel pathway for biomimetic repair [173].

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Different enamel remineralization in vitro by toothpastes containing fluoride or biomimetic CHA microparticles micro-clusters
SEM analysis has been used to investigate the morphology of both demineralized enamel and the features
observed after a remineralization process which utilises in vitro application of toothpastes containing fluoride or CHA micro-clusters constituted of microparticles 100 nm sized [170].
The surfaces of the teeth treated with fluoride (Fig. (9C)) were not consistently changed in respect to thoseof demineralized by ortophosphoric acid (Fig. (9B)). Actually both interprismatic and prismatic enamel structures still appear evident. On the contrary, after treatment of the enamel slabs with a toothpaste containing
synthesized CHA micro-clusters constituted of microparticles 100nm sized the interprismatic and prismaticenamel structures appear to be completely hidden by a thick homogeneous apatitic layer (Fig. (9A)).
The XRD patterns collected on the surface of enamel slabs after treatment with CHA or fluoride tooth-pastes
and water are reported in Figg. (10 b,c,d) respectively and compared with the XRD pattern (Fig. (10a) of CHA
micro-clusters constituted of 100 nm sized microparticles utilized to prepare the used CHA tooth-paste. The
XRD diffraction maxima recorded on the surface of enamel slabs treated with fluoride containing toothpastes appear slightly more sharpened
than those obtained on the enamel
etched slabs brushed only with water.
This observation reveals an increased
crystallinity degree probably due to apartial structural conversion of hydroxyapatite

into

fluoride

substitutedhydroxyapatite. On the contrary, the
XRD pattern obtained on the surface ofenamel slabs brushed with CHA containing tooth-paste shows the broadened
diffraction maxima characteristic of thesynthetic biomimetic CHA, revealing its
presence on the enamel surface. The
CHA not removed by brushing procedures suggests the formation of chemical bonds between the synthetic CHA
micro-clusters constituted of 100 nm
sized microparticles and natural enamel
apatite microparticles. These bonds
allow the formation of a persistent CHA
coating on the enamel surface whosemorphology was detected by SEM analysis.
The surface Ca/P molar ratio determinedby XPS analysis for demineralized enamel slabs before and after in vitro remineralization by brushing with toothpastes
containing fluoride or CHA have been
compared with the Ca/P molar ratio of
CHA micro-clusters constituted of 100
Fig. 9. SEM images of enamel after brushing treatment with: fluoride containing toothpaste (C), enamel surface after application ortophosphoric acid (B)and CHA containing toothpaste (A).

18

nm sized microparticles. The enamel surface Ca/P molar ratio practically does notBrochueProva:Layout 1

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Fig. 10. XRD patterns of enamel after brushing treatment with: water (d), fluoride containing toothpaste (c), CHA microclusters containing
toothpaste (b) and XRD patterns of synthetic CHA microclusters (a). * indicates Al holder diffraction maxima.

change before and after the brushing treatment with tooth pastes containing fluoride. This finding reveals
how the only structural modification of enamel hydroxyapatite induced by fluoride is restricted to a partialhydroxyl group replacement by fluoride ions without affecting appreciably the Ca and phosphate structural
network. On the contrary, enamel slabs after the brushing treatment with the toothpaste containing synthesised CHA micro-clusters of 100 nm sized microparticles the enamel slabs exhibit a surface Ca/P molar ratio
very close to that of the synthetic CHA micro-clusters of 100 nm sized microparticles. The results highlightthat biomimetic nano sized CHA microparticles produce an apatite coating deposition on the enamel surface.
An advantage of the 100 nm sized synthetic building block respect the 20 nm sized in binding on to theenamel surface has been appreciated, but it can be ascribed to a different and more suitable micro-cluster
aggregation.
This coating is much less crystalline than native enamel apatite, and consists of a new apatitic mineral deposition which progressively fills the scratches and pits. On the contrary, the surface remineralisation observedon the specimens treated with fluoride contained in toothpaste, is mainly based on chemical-physical enamel apatite surface modifications rather than on the formation of a new mineral deposition.
The documented CHA biomimetic coating formation, is the first remineralization process corresponding to areal new mineral apatitic deposition in the demineralized area of enamel surface.
Current and future developments
From some decades the teeth health care has been committed to the fluoride effect on hydroxyapatite.
Fluoride interacts with hydroxyapatite promoting the conversion into fluorapatite, hich is less soluble andmore mechanically resistant, but also more brittle than hydroxyapatite.
The action of fluoride on the surface enamel apatite appears ideal to prevent demineralization due to acidfood and drinks and to contrast plaque damage. A lot of scientific works support the fluoride positive action
for teeth health care and consequently a lot of fluoridated oral care products have been patented till nowadays and in some countries drinking water has been enriched with fluoride.
Fluoride is not essential for human growth and its content in the body is not under physiological control.
Adsorbed fluoride is rapidly distributed in the body by circulation and is retained only in calcified tissues. In
the last decades dangerous effects in human health have been ascribed to the daily fluoride amount ingest19

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ed and many studies have put in evidence its high risk, especially for children, to get fluorosis and bone diseases in old people. European Food Safety Authority (EFSA) Scientific Panel has advised about the risk of fluoride intake from ingested oral care products and toothpastes can be fluoridated with a maximum level of1500 mg/Kg. In spite of this knowledge, fluoride continues to be widely used in orthodontia and dental
applications to prevent enamel surface demineralization and represents the main component in toothpastes
claiming remineralization effects. If nowadays fluoride represents the unique agent to contrast caries,
plaques and demineralization we may probably ascribe this situation to the expensive cost of odontoiatricproducts containing hydroxyapatite. In spite of this, hydroxyapatite is commonly considered the main synthetic biomaterial as bone filler and substitute and only few daily use products for teeth health care havebeen patented. Too expensive is the hydroxyapatite cost to be consistently utilised as an active agent in
toothpastes or mouth washes. In fact, almost all the patents previously reported and highlighting the ability of hydroxyapatite to remineralize enamel and dentine, propose different compositions containing calcium and phosphate ions which can produce hydroxyapatite on teeth if mouth pH induces this crystallizationand frequently are obliged to insert a low amount of fluoride in order to promote the apatite crystallisation.
Small amounts of industrial hydroxyapatite are associated to other cheaper components and submitted tomilling to improve hydroxyapatite surface area and reactivity. Only recently the development of nanotechnologies has opened new opportunities in obtaining cheap hydroxyapatite micro-nano particles by the “bottom up” methods.
These hydroxyapatites are surface nanostructured and have higher surface area and consequently higher
reactivity allowing them to bing to enamel and dentine apatite producing on enamel a biomimetic coating,contrasting plaque formation and sealing dentine tubules annulling hypersensibility.
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