WO1999019366A2 - Chelates of chitosans and alkaline-earth metal insoluble salts and the use thereof as medicaments useful in osteogenesis - Google Patents

Abstract

Amorphous or partially crystalline chelates of chitosans, functionalized at the nitrogen with polycarboxylic functions, with alkaline-earth metal insoluble salts. Said chelates are useful for the preparation of medicaments promoting the reparation of bone tissue.

Description

CHELATES OF CHITOSANS AND ALKALINE-EARTH METAL INSOLUBLE SALTS AND THE USE THEREOF AS MEDICAMENTS USEFUL IN OSTEOGENESIS

The present invention relates to chelates of chitosans with inorganic salts, in particular to amorphous or partially crystalline chelates consisting of chitosans functionalized at the nitrogen with polycarboxylic functions, linked to alkaline-earth metal insoluble salts, suitable for the preparation of medicaments useful to promote the reparative process of bone matrix and its mineralisation.

The mineralised tissues of vertebrates contain crystals of carbonated hydroxyapatite , which have grown in an organized way inside a collagen fibril structure. Vesicles containing calcium ions and phosphate ions are formed in the matrix, they come close to the collagen fibrils and give origin to crystals growing in the form of plates, 35x8x1.5 nm (tendon) [Ertε et al . , 1994] or 50x25x3 (bone) [Zhang and Gonsalveε, 1995]. Parent compounds are also in equilibrium with complexing agents .

Biological hydroxyapatiteε exhibit low crystallinity and lack εtoichiometry due to the presence of ions abεorbed or included in the cryεtal lattice [Bigi et al. , 1994] .

The inorganic component of theεe tiεεueε is formed in the preεence of various substances exerting inhibiting actions and structural alterations; for instance, hydroxyapatite is formed according to the reaction: 2CaHP0₄ + 2 Ca₄(P0₄)₂0 >Ca₁₀ ( P0₄ ) ₆ ( OH ) ₂ at 38°C which iε effected by the influences of macromolecular species, e.g. albumin, ionic species, e.g. the hydrogen carbonate ion, and other species which produce important alterations, such as hydroxyapatite carbonation [Martin and Brown, 1994] .

Various recipes for the preparation of hydroxyapatite-based bone cements have been described by

Driesεenε et al., 1994, Akai, 1994 and Ito et al., 1994. However, the treatment of bone leεions with said cements iε relatively naive, becauεe hydroxyapatite iε the ultimate product of the mineraliεation process in vivo . The artificial hydroxyapatite granuleε are not valid εurrogates of the nanocryεtals generated in vivo: on the contrary, they εlow down the cellular proliferation and can be conεidered foreign bodies. Porouε hydroxyapatiteε and coated hydroxyapatites were developed to obviate to these drawbackε [Eggli et al., 1987].

Variouε cementε containing chitoεans have been propoεed by Sumita, 1988, who mixed tricalcium phoεphate and tetracalcium phoεphate with chitoεan acid εolutions: the reεulting material hardenε within 12 minutes and the hardening iε believed to take place following formation of hydroxyapatite [Lacont et al., 1996]. Takechi et al., 1996 alεo suggeεt chitoεan or alginate to obtain a time- stable cement and aεcribe the cement stability to the decrease in liquid-permeability carried out by the biopolymer. Analogously Ito, 1991, mixes hydroxyapatite with ZnO, CaO and chitosan. Iεhhii mixeε Ca₃(P0₄)₂ with chitosan. Hydroxyapatite capability of interacting with biopoly erε iε well documented in the chromatography field.

Wherever a cement is not εtrictly neceεεary for the medication of a bone defect, materials conceived in a totally different way might be more desirable. Bones being subject to a continuouε reεtructuring by osteoblasts, resorbable hydroxyapatites have been developed as compounds that oεteoblaεtε can restructure. Such hydroxyapatiteε εhould be inorganic compoundε compriεing biopolymerε intercalated in the crystal lattice [Meεεerε ith and Stupp, 1992]. However, εuch materials are not at present available. Lohman, 1993, discuεεeε the preparation of modified chitoεanε, giving εome generic information on the aεpect of NaCl, CaS0₄ and CaC0₃ cryεtalε, obtained in the preεence of biopolymerε, but he doeε not characteriεe the resulting materials .

Chitin is often found in combination with CaC0₃ in insectε and arthropods.

While chitoεan and many of itε derivatives have chelating ability towardε tranεition metal ionε but not for calcium, [Muzzarelli and Tubertini, 1969], chitoεanε capable of chelating calcium have been prepared by functionalizing chitoεan with polycarboxylic groupε [Muzzarelli and Delben, 1989]. For example, uεing the correεponding keto acidε and chitosan, Muzzarelli and Zattoni, 1986, prepared glutamate glucan and aspartate glucan, which have widely been characterized by Chieεsi et al. [1992]. Furthermore, Rinaudo et al. [1994] and Muzzarelli et al. [1985] have prepared highly carboxymethylated chitoεans. Said chitosans have been described for the separation of metal ions and for research on their εtructure, but nothing is known in termε of their effect on the cryεtallization of εparingly εoluble εaltε. Gruber [1996] εuggestε, for example, the reaction of cis-epoxysuccinic acid (oxirane-dicarboxylic acid) with a chitosan salt, to give N-[ ( 3 ' -hydroxy-2 ' , 3 ' -dicarboxy )ethyl]chitosan for cosmetic purposes, without discussing its chelating capacity. On the other hand, Inoue et al. [1996] syntheεized a class of chitosanε carrying chelating EDTA-type functional groups and uεed them for the separation of lanthanides and other metal ions. Inoue et al. [1995] discuεε the εelectivity of εaid chitoεans towards some transition metals. Analogouεly, Ina and Nakamura [1992] propoεed carboxymethylated chitoεanε for the preparation of detergents. No Author who described carboxylated chitosans ever disclosed bioinorganic compounds in terms of novel compoεitions of the matter; on the contrary, they only conεidered aεpectε of analytic chemiεtry or preparation of mixtures, see Menon et al. [1995], Rinaud et al. [1994] and Guibal et al. [1995] .

The osteoinductive, oεteoconductive , reεorption capacitieε of chitin- and chitoεan- baεed materials have already been reported by Muzzarelli et al. [1993] and Muzzarelli [1996]. Such capacities are not present in alginates, therefore no relationεhips between alginate- Ca chelates and the modified chitosan - alkaline-earth metal chelates of the invention could be foreseen.

It has now surprisingly been found that the well- known precipitation reactions with alkaline-earth metals, in particular calcium, when carried out in aqueouε medium in the preεence of a highly carboxylated chitosan give amorphous or only partially cryεtalline compoundε, incorporating a biopolymer, and which can, in some cases, be isolated as water soluble materials. Summary of the invention

The present invention relateε to amorphous or partially crystalline chelates consiεting of highly carboxylated chitosanε linked to alkaline-earth metal inεoluble εaltε. A further object of the preεent invention iε a proceεs for the preparation of said chelates.

A still further object of the preεent invention is the uεe of εaid chelateε for the preparation of medicamentε useful in human and animal medicine, together with pharmaceutical compositions containing said chelates as active ingredients.

The present invention also relates to the use of said chelateε for the coεmetic treatment of human body, as well as the cosmetic co positionε containing them. The advantages provided by the present invention basically derive from:

1) Characteristics of homogeneity, solubility, malleability and hydrophilicity of the bioinorganic compounds isolated in the form of solid materials. 2) No or very poor crystallinity characteristics.

3) Biocompatibility , biodegradability and complete resorption.

4) Oεteoinductive, oεteointegration and accelerated mineraliεation of the bone medicated with εaid bioinorganic materials.

5) Simple preparation and iεolation processeε. From the activity point of view, therefore, the product has both an organic and an inorganic component: the first one provides a carrier for the adhesion, migration and proliferation of the tissue cells and a carrier for the correct regeneration of the extracellular bone matrix, the second one acts as a nucleation medium for the onset of the mineralisation procesε and aε a carrier for the ex. novo formation of hydroxyapatite. Thiε promoteε the healing proceεε and the regeneration when thiε cannot occur εpontaneouεly , such as iε the caεe with defects of wide size.

The present invention will be disclosed in further detail by the following Examples and Figureε, in which:

Figure 1 shows the X-ray diffraction spectra for brushite (A), and for εome chelateε of the present invention (B) and (C), as further illustrated below, in particular for DCMC-Ca-P chelateε;

Figure 2 εhowε the X-ray diffraction εpectra for some chelates of the preεent invention (A) and (B), in particular for DCMC-Ca-F chelateε.

According to the present invention, highly carboxylated chitosans (in the following referred to as functionalized chitosanε, for εake of εhortneεε) are known in literature and the preparation thereof iε diεclosed in a number of publications. According to a preferred embodiment of the invention, the functionalized chitosan is selected from dicarboxymethyl chitosan (obtained from glyoxylic acid), glutamate glucan (obtained from ketoglutaric acid), aspartate glucan (obtained from oxalacetic acid) and N-[(3'- hydroxy-2 ' ,3 ' -dicarboxy )ethyl]chitosan (obtained from oxiranedicarboxylic acid).

The present invention is not limited to specific alkaline-earth metal insoluble saltε. Examples of said salts are phoεphate, sulfate, oxalate, carbonate, hydrogen carbonate, fluoride of one of the various alkaline-earth metal cations.

In a preferred embodiment, said salts are calcium saltε, particularly calcium phosphate.

A most preferred chelate is that of calcium phosphate with a derivatized chitoεan in a 1:1 weight ratio.

The process for the preparation of the chelateε of the present invention comprises the following steps: disεolution of the functionalized chitoεan in a εolution of a soluble salt, preferably the sodium salt, of the desired alkaline-earth metal anion; addition of a soluble salt of the desired alkaline- earth metal anion in equimolar amounts, to give the chelate with the alkaline-earth metal insoluble salt, and subsequent isolation.

Alternatively, the process according to the present invention comprises mixing carboxylated chitosan with an insoluble salt of the deεired alkaline-earth εalt.

In the exemplary caεe of a calcium chelate, the carboxylated chitoεan is dissolved in a solution of the selected sodium salt and a soluble calcium salt is added in equimolar amounts, to form an insoluble calcium salt. ■

The reaction conditions will be selected by those skilled in the art, depending on the functionalized chitosan and the final alkaline-earth metal inεoluble εalt. A most preferred embodiment of the invention provides the chelate of dicarboxymethyl chitosan (in the following DCMC) with an inεoluble calcium salt.

Table A summarizeε macroεcopic observations for a number of anions in water at the specified pH values, when reacted with calcium ions, in the presence of DCMC.

TABLE A

Sodium salt Final pH Aεpect of the syεtem with DCMC with no DCMC

phosphate 6.1-6.2 clear, homogeneous precipitate sulfate 5.9-6.7 turbid clear, homogen. oxalate 5.8-6.6 turbid precipitate carbonate 8.3-9.0 turbid, no precip. precipitate hydrogen carbonate 7.2-9.0 turbid clear fluoride 6.2-6.9 precipitate with turbid clear supernatant

For selected molar ratios, specified below, it is posεible to observe that, in the presence of DCMC, calcium phosphate does not precipitate. This behavior differs from the regular behaviors of calcium salts in the absence of the biopolymer.

When DCMC iε reacted with calcium ionε , the reaction reεults in products of various aspect, depending on the Ca/DCMC molar ratio, as shown in table B below.

TABLE B CALCIUM MOLAR RATIO OBSERVATIONS

(μ mols) Ca/DCMC

1.5 0.0125 No precipitate

2.0 0.0156 No precipitate

2.6 0.0208 Turbidity

5.2 0.0400 Precipitate

7.7 0.0588 Gelling precipitate

26 0.2000 Precipitate

52 0.4000 Non-homogeneous precipitate

77 0.5882 Non-homogeneous precipitate

128 Non-homogeneouε precipitate

Note:

DCMC concentration: 0.56% (128 μmols) Ca acetate concentration: 1.0% Temperature 20"

In practice precipitation occurs for high ratios, whereaε no precipitate iε obεerved for low ratios; but for ratio 0.0588 a rigid gel forms, that takeε the εhape of the container and can be sliced with a knife. The

DCMC-Ca gel disεolveε upon contact with a diεodium orthophosphate εolution, yielding a clear εolution, with no pH changes observed (5.5).

The most favourable conditions for the formation of a water-soluble DCMC-calcium phosphate chelate (DCMC-Ca-

P) are illustrated in the following table C.

TABLE C

DCMC Ca/DCMC ratio Notes μ ols grams mgrams molar weight

DCMC 0.56% so: . DCMC

2.6 0.1 0.56 51 9 precipit. similar to the

5.1 0.2 1.1 25 4.7 control

7.7 0.3 1.7 17 3 id.

10.0 0.4 2.2 13 2.4 id.

13.0 0.5 2.8 10 1.9 id.

15.0 0.6 3.3 7 1.6 id.

31 1.2 6.7 4 0.7 id.

54 2.1 11.8 2.4 0.4 clear

79 3.1 17.4 1.6 0.2 precipitate

94 3.7 20.7 1.4 0.25 id.

110 4.4 24.6 1.2 0.12 id.

130 5.0 28 1 0.18 id.

It can be observed that, for equimolar amounts of diεodium hydrogen orthophoεphate and calcium acetate, a

Ca/DCMC 2.4 molar ratio giveε a homogeneouε εystem, from which a water-soluble DCMC-Ca-P chelate can be recovered by freeze-drying. Thiε product is soluble in a wide range of pH, but the polymer can easily be isolated from mother liquors with ammonium hydroxide (pH 8).

X-ray diffraction spectrometry.

As illustrated in Figure 1, spectra recorded for compoundε deεcribed at lineε 1-5 of table C, dried at

40 °C, εhowed a marked depression of the peaks typical of brushite (Figure 1A) at 20 values of 11.68, 20.96 and

29.28, which are, on the contrary, very intense in the control precipitate under the same experimental conditions and which correspond to the values of the crystallographic tables. Of the other peaks, only that at 4.64 20 is further affected by the DCMC concentration (Figure IB, Table C, sample of line 5).

The spectra showed that even small a ountε of DCMC in the precipitation medium lead to almoεt a orphouε DCMC-

Na HP0 .2H₂0 combinations, in which no diffractometric peaks of brushite are present (Figure 1C, Table C, sample of line 8 ) .

Reflectivity Infrared Spectrometry. Analysis were carried out on samples prepared as follows:

DCMC: a 0.4% DCMC solution was treated with glacial acetic acid diluted 1:3 to pH 6.09. The reεulting solution was treated with acetone and the precipitate waε obtained by centrifugation, then freeze-dried. The reεulting product waε ground with KBr for analysis.

DCMC-P-Ca: was prepared treating a 0.4% DCMC solution (75.2 g) with Na₂HPO₄.2H₂₀ (1%, 18 ml), then with calcium acetate (1%, 21 ml). Part of this solution (20 ml) was treated with acetone and centrifuged. The freeze-dried compound was ground with KBr.

The spectra showed that the reaction with calcium phosphate induced a shift of the peaks from 1640 to 1590 cm^~l and the lowering of the peaks at 1400 and 1150 cm^" . Remarkable iε also the lowering of the two peaks at

540 and 470 cm^-1.

Elemental analysis

Preliminary elemental analyεiε on the DCMC εample εhowed that the chemical functionalization introduces carbon and oxygen into the polysaccharide with a reεulting N/C ratio for DCMC (0.115) lower than for plain chitoεan (0.182), as expected following carboxymethylation. The sum N+C+H for DCMC is lower than 40%, in that sodium and the increased oxygen should be considered, while for plain chitosan it is close to 60

%.

In the derivative containing calcium phosphate, the

N percent drops to less than one half (1.66) of the corresponding value for DCMC (3.53) and therefore the organic/inorganic ratio in the chelate is about 1:1 by weight . TABLE D

Elemental analyεiε data on chitoεan with a 0.20 acetylation degree and on itε derivatives.

percent chitosan DCMC DCMC-P-Ca gr .ac. 0. .20 2496

N 8.26 3.53 1.66

C 44.71 30.59 21.68

H 4.90 3.51

N/C 0.182 0.115 0.076

Behavior of other sparingly soluble calcium salts.

The other sparingly soluble calcium salts tested as described above are: sulfate, oxalate, carbonate, hydrogen carbonate and fluoride. No εolubilization phenomena εimilar to that with the phoεphate εalt have been observed, always obtaining a precipitate upon mixing the saltε. On the contrary, amorphouε or poorly cryεtalline εaltε formed analogouεly. In particular, Figure 2 reports the X-ray diffraction spectra for the DCMC-calcium fluoride derivative (abbreviated DCMC-F- Ca). The precipitation of calcium fluoride starting from calcium acetate and sodium fluoride in the preεence of DCMC reεultε in productε which, once dried at 55°C, become hard and yellowish, especially those iεolated from media with higher DCMC content. In the X-ray diffraction spectra for these compoundε, the characteriεtic peak for calcium fluoride <111> at 28,3 20 εhowed intensity inversely proportional to the DCMC content.

The chelates of the present invention promote the mineralisation of newly formed bone tisεue, and are therefore uεeful for the preparation of medicamentε for the treatment of pathological conditionε of bone tiεsue.

A study waε carried out on εheep to evaluate the activity of the chelateε of the invention. Sheep were εelected in view of histologic, physico-mechanical and phyεiopathological εimilarity to human femurε.

The day before εurgery, 100 mg/kg of Cefazolin

(Cefamezin) were adminiεtered to the animalε. The animalε underwent εurgery after εtarving for 12 h. Following a premedication with ketamine (10 mg/Kg) and xylazine (0.2 mg/kg) general anaeεthesia waε induced with thipethane-natrium (8 mg/kg, 2.5 % solution) and maintained with a gas mixture of 0.5-1.0 % Halotane and N₂0+0₂ (1:1) in automatic ventilation. A bone defect was created in the femural epiphyseε by means of a 6 mm drill. During surgery the drill holes were carefully rinsed with 0.9 % NaCl solutionε and cleaned out in order to remove abraded particles, reduce drilling temperature and avoid bone necrosis. In the right leg the hole was completely filled with imidazolyl-chitosan or DCMC-Ca-P-chitoεan, while in the other one it waε left open to serve as control. The wounds were sutured atraumatically in two layers and disinfected. Animals were submitted to antibiotic therapy (25 mg/]cg cefazolin pro die) for 7 days, and were allowed to bear weights as tolerated. Neither intra- and post -operative nor general and local septic complications were observed. Animals were sacrificed 40 and 60 days after surgery. Femurs were removed and εawn to produce fragments for histologic examination. In sheep medicated with DCMC-Ca-P, macroscopic analysiε evidenced an irregular rimmed area εmaller than the εurgical defect, filled with a tiεsue without histoarchitectural characteriεtic of bone tissue. In control femurs the hole had not changed much in shape and size since surgery and the area lacked in bone tissue. Microscopic analysis of treated legs clearly showed the difference between the reparative- reconεtitutive bone tiεεue with or without chitoεan. Firεt of all the number of cellε were higher in the treated legε than in the control oneε, and their large εize and εtar-shape were suggestive for activation. However, the most important factor waε the preεence of a wide oεteogenic reaction (Figs, a, b) moving from the rim of the surgical lesion toward the centre. Vascular endothelial cells synthesize and secrete an array of soluble mediators either conεtitutively or in reεponεe to induction εti uli εuch as injury or inflammation. Among these, it is important to mention growth factors and cytokines: fibroblaεt growth factor (FGF), interleukin-1 (IL-1), interleukin-6 (IL-6), colony εimulating factorε (SCUFFS) of the G, GM and M subtypes, arachidonic acid, prostacyclin, and small peptides like endotelin-1. These regulatory compounds have been seen, in other εtudies, to control the recruitment, proliferation, differentiation, functioning, and/or survival of various cells including bone-forming osteoblasts and bone- degrading oεteoclastε. Laεtly, endothelial cell releaεe of other εhort- live meεεenger molecules, namely reactive oxygen species and nitric oxide (NO), may help to control osteoclast activity. In fact, activated oxygen species seem to enhance resorption, whereas NO has been εhown to interfere with osteoclast bone resorption and, furthermore, inhibitors of NO synthetase potentiate osteoclaεts bone-resorption. The results illustrated in the present invention evidence the important role of

DCM-chitosan in stimulating bone tisεue reconstitution and suggest that their action can be potentiated by chelation of calcium phosphate.

In a further embodiment of the invention, the chelates are useful in dentistry.

The avulsion of a dental unit and a cyεtiε in a 15- year old patient was performed: the space left was filled with freeze-dried DCMC-CaP. Radiographic observationε εhowed precocious formation of osseous trabeculae 15 days a.s.

As far as the industrial applicationε of the invention are concerned, an aspect thereof are pharmaceutical or cosmetic compositionε containing an effective amount of one or more chelateε of the invention as active ingredients, in admixture with conventional carriers and excipients. Said compoεitions are prepared in forms and according to procedures well- known to those skilled in the art, as described, for example, in "Remington's Pharmaceutical Sciences Handbook", Mack Publishing Company, New York, U.S.A.

Dosages and poεology will be determined by the physician, depending on the type of the pathology to be treated, as well as on the conditions of the patient and any other useful considerationε . A further, εpecific embodiment of the preεent invention provideε medical articleε in the formε of freeze-dried aterialε, powders, films, membranes, solutionε, gels and malleable pastes. A further preferred embodiment provides the chelateε of the invention aε active ingredientε in compoεitionε for εtrengthening teeth enamel, εuch aε tooth-paεteε , paεtes and gels.

The chelates of the invention are also useful for the treatment of hair and can suitably be formulated in cosmetic compositions.

The following examples further illustrate the invention.

Example 1: Preparation of DCMC-Ca chelate. A series of solutions are prepared, so as to have a DCMC constant content and a calcium acetate increasing content, pH being 5.5. When the Ca/DCMC molar ratio is lower than 0.02, no precipitate is observed, whereas at 0.2 - 0.5 ratios precipitation occurε. For intermediate valueε 0.06-0.20 a rigid gel formε, that takeε the εhape of the container and can be εliced with a knife. The gel rediεεolveε when placed in a phosphate εolution, yielding a clear solution. For values from 0.4 to 1.0 a non-homogeneous precipitate is formed. Example 2: Study of the DCMC-Ca-P εystem.

When solutions containing both DCMC (at variable concentration) and Na HP0₄.2aq (at constant concentration) are treated with calcium acetate (at constant concentration), precipitation takes place in all cases, except for Ca/DCMC molar ratios close to 2.4- 4.2, as εhown in table C. Example 3: Preparation of the bioinorganic compound

DCMC-Ca-P .

The aqueous solution of DCMC (0.48%, 220 g) and of Na₂HP0₄.2aq (1.0%, 67 ml) iε mixed with the calcium acetate one (1.0%, 72 ml). Referred to 1 g of DCMC (dry weight), the amountε are 0.6 g of εodium salt and 0.7 of calcium acetate. The resulting εolution is dialyzed and freeze-dried, to give a spongy material which becomes a gel when mixed with water or saline. Example 4: Preparation of the bioinorganic compound DCMC-Ca-F .

The formation of the chelate DCMC-Ca-F from NaF and calcium acetate solutionε in the presence of DCMC yields products that, once dried, become hard and yellowish. In the X-ray diffraction spectra, the single (111) peak at 28.34 20 typical for CaF showed intenεity inverεely proportional to the DCMC concentration.

Example 5: Preparation of the bioinorganic compound DCMC-Ca-carbonate . A conεtant amount of calcium acetate (1.0% solution, 3.2 ml = 164 micromols = 6.55 mg Ca) iε added to a mixture of DCMC (0.48% at pH 6 in variable amount, with 0.1 N ammonia) and of εodium carbonate (1.0%, 1.7 ml = 164 micromolε = 0.017 g). No formation of precipitate is obεerved, although the solutions remain turbid, whereas the control (no DCMC) gives a precipitate . Bibliography

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Claims

1. Amorphous or partially crystalline chelates conεisting of chitosanε, functionalized at the nitrogen with polycarboxylic functions, linked to alkaline-earth metal insoluble salts.

2. Chelates according to claim 1, wherein εaid insoluble saltε are calcium εaltε.

3. Chelates according to claim 2, wherein εaid inεoluble εalt iε calcium phoεphate.

4. Chelates according to claim 3, wherein the weight ratio of calcium phosphate to functionalized chitoεan is 1:1.

5. A procesε for the preparation of chelateε of claims 1-4, which comprises the following steps: a) mixing of a functionalized chitosan with a εoluble salt of the desired alkaline-earth metal anion; b) addition of a soluble salt of the desired alkaline- earth metal to form the chelate with the alkaline- earth metal insoluble salt; c) isolation of the chelate.

6. A process according to claim 5, wherein the functionalized chitosan is selected from: dicarboxymethyl chitosan, glutamate glucan, aεpartate glucan, N-[ ( 3 ' -hydroxy-2 ' , 3 ' -dicarboxy )ethyl]chitosan.

7. A procesε according to claim 5 or 6, wherein the salt of step a) is a sodium salt selected from: phosphate, sulfate, oxalate, carbonate, hydrogen carbonate, fluoride.

8. A process for the preparation of the chelates of claims 1-4, which compriseε mixing the functionalized chitosan with an insoluble salt of the desired alkaline- earth metal.

9. A process according to any one of claims 5-7 or to claim 8, wherein the molar ratio of alkaline-earth metal salt to functionalized chitoεan iε 1:3.

10. The uεe of the chelates of claims 1-4 as carriers for medicaments or growth factors.

11. The use of the chelates of claims 1-4 for the preparation of a medicament useful to promote the restructuring procesε of the bone matrix and for the mineraliεation thereof.

12. Pharmaceutical or coεmetic compoεitionε comprising an effective amount of at least one chelate of claims 1- 4 as active ingredient, together with conventional carriers and/or excipients.

13. Medical and coεmetic articleε containing the chelates of claims 1-4.

14. Medical articles according to claim 13, in the form of freeze-dried materials, powders, films, membranes, solutions, gels and malleable pastes.