WO2009077210A1

WO2009077210A1 - Monetite matrices and their application in bone regeneration

Info

Publication number: WO2009077210A1
Application number: PCT/EP2008/058694
Authority: WO
Inventors: Faleh TAMIMI MARIÑO; Enrique Lopez Cabarcos; Jorge Rubio Retama; Jesus Torres Garcia
Original assignee: Universidad Complutense De Madrid; Universidad Rey Juan Carlos
Priority date: 2007-10-15
Filing date: 2008-07-04
Publication date: 2009-06-25

Abstract

The present invention refers to synthetic monetite matrices with different degrees of porosity and their application in bone regeneration. These matrices are produced from a brushite cement resulting from mixing a solid phase containing a basic calcium phosphate and an acid calcium phosphate, and the incorporation of an aqueous phase. The resulting solid brushite matrix is subsequently converted into monetite by heat treatment. These synthetic monetite matrices can be made as granules or preformed shapes with different porosities, and can be made to incorporate bioactive agents that promote bone formation.

Description

MONETITE MATRICES AND THEIR APPLICATION IN BONE REGENERATION

INVENTION FIELD

This invention belongs to the technical sector of biomaterials, more precisely, to the field of calcium phosphates used in bone regeneration. The synthetic monetite matrices of the present invention result in osteoinductive materials that can be used in dentistry maxillofacial surgery, periodontal and other applications were bone regeneration is needed.

BACKGROUND

Loss of bone mass and quality is a serious health problem that is aggravated in patients with old age. In dentistry treatments it is common that surgical interventions result in the loss of bone that may in turn lead to other complications and pathologies. As an example, this happens in alveolar resorption following a tooth extraction, and also in periodontal disease. In addition, in traumatology as well as in neurosurgery, bone loss represents a serious health problem that can even result in death of the patient.

For nearly a century biomaterials have been used to repair or replace bone segments of the skeletal system. Use of autologous bone, that is from the patient, is a common procedure used to refill bone cavities and for surgical reconstruction. However, there is a limited source of bone and this imposes on the patient an additional trauma to obtain the graft material. Another option is that of heterologous grafts from donors. However these show a slower bone neoformation, inferior osteogenic capacity, higher bone resorption and immune responses, reduced revascularization and a higher risk of transmission of pathogenic agents.

Research into new biomaterials for bone repair aims at reducing as much as possible the need for bone grafts, aiming for artificial succedanea that is reabsorbed in time and/or integrates with adjacent bone, and that also serves as a fixture for osteoporotic fractures. The mechanical properties of this succedaneum of bone mineral should be as close as possible to that of spongy bone. The material must also aid in providing fracture stability and be sufficiently robust to reduce the time for which immobilisation or external support is necessary.

The succedaneous material must be biodegradable, biocompatible and osteoinductor, that is, it must attract mesenchymal cells located near the implant and favour their differentiation into osteoblasts while also acting as a mould for the formation of new bone.

Calcium phosphates are of special interest in bone regeneration as they are similar to the mineral phase of natural bone and they are susceptible to osseous reshaping and resorption. The most commonly used calcium phosphate matrices include hydroxyapatite, tricalcium phosphate, or brushite. These materials can be administrated in form of cement pastes, implantable solids, powders or granular formulations.

In the context of cements that set after their implantation it is worth mentioning those that result in the formation of hydroxyapatite as Norian®, or those that result in brushite as Chronoss Inject®. Formulation in the form of cements allows for the administration in locations of difficult access by means of high calibre syringes. However, these cements may result in exothermic reactions during setting, pH changes, or formation of crystalline structures that do not favour osteoinduction.

As an alternative to the use of cements that set after implant, it is worth mentioning the administration of osteoinductive materials in the form o solid matrices in the form of powder, granules or as preformed implants.

Examples of preformed implants include, for example, patent US- 4610692, which describes a tricalcium phosphate preformed implant soaked with antibiotics. Furthermore, patents US-5866155, US-6203574 or US-2005209704 describe processes where the preconfigured implant is formulated as the product of the fusion of calcium phosphate granules with other polymers to provide a shape to the implant. However, the incorporation of large masses and more so if they contain polymeric materials, may result in local pH alterations and a negative effect in the osteogenic capacity of the implant.

As an alternative, powder or granulated formulations permit an easier colonization of the implanted material by osteogenic cells, and the adaptation to corporal cavities of irregular shape. Examples of the direct use of bovine bone granulates are represented by BioOss® an Orthoss® for use in dentistry. However, use of these preparations obtained from biological material represents problems of possible contamination with infectious agents and require strict quality control. In order to avoid these problems other natural matrices in the form of granules have been developed. Examples of hydroxyapatite granules of coralline origin include Interpore200® and InterporeδOO®. A preferred approximation is the use of synthetic matrices. For example, Gen-ox® o Engipore® incorporate granulates of pure synthetic hydroxyapatite. Other commercial examples of granular synthetic matrices are Chronos® or Cerasorb® made of tricalcium phosphate beta. The latter is marketed in the form of particles of different sizes, from 150 to 2000 μm as needed, which are applied for alveolar regeneration after mixing with blood from the patient. Another similar product, Bi-Ostetic® is formed by 1-2 mm particles of a mixture of hydroxyapatite and tricalcium phosphate. Yet, Collagraft® is other granulated material of hydroxyapatite and calcium tri-phosphate additionally made to contain collagen. Other osteoinductive synthetic materials incorporated in commercial products as Calmatrix® include calcium sulphate.

In the area of materials of increasing interest in bone regeneration is the calcium phosphate dihydrate or "brushite" [CaHPO₄^HbO]. Some of inventors of the present invention have described the advantageous use of brushite in granulated form for bone regeneration (Tamimi FM et al. 2006 J. Clin Periodontology; 33: 922-928). Patent US-3913229 also makes use, among others, of brushite for bone regeneration, although it erroneously identifies de compound as CaHPO₄. The document describes the use of small crystals or preferably granules resulting from pulverization of solid matrices formed by a sintering process that generally implies temperatures between 600 and 1500⁰C, and their preferred administration in the form of a paste. The priority date of the patent is previous to the development of brushite cements (LeGeros et al. 1982 J. Dental Res. 61:343. or Brown WE and Chow LC. 1983 J. Dental Res. 62: 672) and therefore it can be deduced that the materials used are of natural origin or, if it is the case, by conversion of a natural brushite. The patent is only reduced to practice with tri calcium phosphate and does not refer to the material in any of its Examples as brushite or monetite.

Dehydrated calcium phosphate or "monetite" [CaHPO₄] is a material significantly different of brushite, which can be found as a mineral in the nature or that can be synthesized by dehydration of brushite mineral.

Monetite in the form of powder mixed with blood has also been the subject of bone regeneration studies (Getter L et al. 1972 J. Oral Surg. 30: 263- 268). The publication describes the utilization of a monetite powder of undetermined size or crystalline structure, and does not mention the origin of the monetite used. As for Patent US-3913229, the publication date is previous to the development of brushite cements and it can therefore be inferred that the monetite used is of natural origin or by conversion of a natural brushite. Monetite has also been proposed as an additive in hydroxyapatite cements (Barralet et al. 2004 Biomaterials, 25(11 ): 2187- 2195). In WO98/58602 the use of protein solutions in the administration of small hydroxyapatite crystals and others calcium phosphates including monetite is described. The patent describes the problem in the administration of small crystals and is restricted to be administration in combination with 10/30% protein. The document does not mention possible granular forms of monetite. There are also documents such as patent US2005209704 that claim the incorporation of monetite to biodegradable polymers for the formulation of preformed implants. In general, the incorporation of biodegradable polymers such as those of lactic or glycolic acid, result in matrices that in their degradation generate an acid pH which is detrimental in the induction of bone regeneration.

It is known that sterilization of brushite by means of autoclave results in its transformation into monetite (Dorozhkin SV. et al. 2000 J. of Materials Science: Materials in Medicine 11(12)). These transformation processes are frequently associated to chemical transformations, generation of large crystals and a detrimental effect over its osteoinductive capacity. In Tas AC and Bhaduri SB (2004) J. of the American Ceramic Society 87(12): 2195-2200, the conversion reaction from monetite to brushite is described in the context of the production of brushite as the preferred material for bone regeneration.

Taking into consideration the state of the art, it would be desirable to have synthetic osteoinductive matrices which can be easily produced for their application in bone regeneration.

INVENTION SUMMARY

The present invention incorporates a new synthetic biomaterial for bone regeneration, a method for their production, and its application in dentistry, maxillofacial surgery, periodoncy, traumatology surgery, and other indications in which bone regeneration is necessary. The material is based on biocompatible, biodegradable and osteoconductive elements.

In particular, the present invention incorporates synthetic monetite [CaHPO₄] matrices with different degrees of porosity, in the form of granules or preformed blocks, produced by means of setting of a brushite [CaHPO₄-2H₂O] cement and subsequent heat treatment. The matrices can be made in moulds with the desired size and shape, modified by erosion, or pulverisation. Furthermore, the matrices can incorporate other bioactive agents that promote bone regeneration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention refers to a new synthetic biomaterial for bone regeneration and tissue engineering in dental surgery and traumatology based on monetite [CaHPO₄] solids in the form of porous granules or matrices generated by the conversion of a synthetic material of brushite [CaHPO₄-2H₂O]. The material is easily applied to the patient and has demonstrated to be highly osteoinductive. Furthermore, the material in its most simple form avoids the use of other polymers and reagents, and can be produced in a sterile in a granular form or as preformed matrices.

Therefore, a first aspect is referred to a synthetic monetite matrix (CaHPO₄) characterised by having a crystal size of between 0.5 and 25 microns. Preferably presented in the form of granules between 0.2 and 2 mm in diameter and more preferably is presented in the form of blocks.

Other preferred embodiment of this invention is directed to the matrix described above with an induced porosity, between 0 and 20% and more preferably with a total porosity between 35-60%.

The production method for these matrices incorporates the following steps.

A first step in which a brushite cement composed of a liquid phase and a solid powder phase is allowed to set. The solid phase comprises a first component that includes a basic calcium phosphate, as for example [alpha]- tricalcium phosphate, [beta]-tricalcium phosphate, hydroxyapatite, tetracalcium phosphate, octacalcium phosphate and/or calcium oxide; and a second component that includes an acid calcium phosphate, as for example monocalcium phosphate unhydrate or monocalcium phosphate monohydrate.

The solid component can also incorporate additives to control the setting time of the cement so that, at 25⁰C, the setting time is between 1 and 300 minutes, and preferably between 1 and 20 minutes. These additives include, with out restriction, sodium pyrophosphate, calcium pyrophosphate, potassium pyrophosphate, strontium chloride, strontium renalate, strontium pyrophosphate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, sodium phosphocitrate, potassium phosphor citrate, sodium sulphate, potassium sulphate, calcium sulphate hemihydrate, sodium dihydrogen pyrophosphate, magnesium sulphate, sodium bisphosphonate, potassium bisphosphonate, chondroitin 4- sulphate, chondroitin 6-sulphate, glycolic acid, sodium glycolate, and/or calcium glycolate. Barium salts, such as barium titanate, sulfate, or fluoride, can be incorporated as additives to control the setting time of the cement, and can improve radiopacity and the biological response of the material.

The liquid phase can incorporate an acid that includes, with out restriction, phosphoric, glycolic, tartaric, citric, succinic, malic, lactic, hydrochloric and/or sulphuric acid at a concentration between 0.5 y 3.5 M.

The components of the cement can also include additives that increase the porosity of the resulting matrix through the liberation of gas during the setting process, as for example, and with out restriction, calcium carbonate, calcium bicarbonate, sodium bicarbonate, or hydrogen peroxide, or other salts. Furthermore, porosity of the resulting matrix can also be increased by means of the incorporation to the cement of additives that following setting and dissolution result in the formation of pores. Examples of these additives include, with out restriction, organic or inorganic salts, sugars, sugar alcohols, aminoacids, proteins, polysaccharides or polymers.

The cement may also incorporate additives to control the rheology of the cement. These additives comprise, with out limitation, chondroitin A- sulfate, chondroitin 6-sulfate, a silica gel, a silica gel with chondroitin 4- sulfate, a silica gel with chondroitin 6-sulfate, strontium chloride, strontium renalate, and any salt containing strontium, sodium pyrophosphate calcium pyrophosphate and or any salt or acid containing pyrophosphate groups. Biocompatible agents such as, and with out restriction, collagen, chitosan, albumin, fibronectin, hyaluronic acid, hyaluronate salts, dextran, alginate, xanthan gum or celluloses, can also be incorporated to control the rheology of the cement. Specially, it can incorporate high molecular weight hyaluronic acid.

The liquid phase can also contain additives to control the cohesion of the cement which can be selected between chondroitin 4-sulfate, chondroitin

6-sulfate, silica gel, or a combination of the silica gel with either of the previous two. The concentration of the chondroitin 4-sulfate and the chondroitin 6-sulfate in the liquid phase is between 1 and 6% while the concentration of the silica gel can be between 1 and 15g/L.

For the generation of the cement, the powder components are mixed with the liquid phase in a proportion powder (grams) to liquid (ml) that can be between 0.2 and 10 g/ml, preferably between 1 and 3 g/ml. When the solid and liquid phase are mixed a paste is formed that subsequently sets and solidifies to form a material which consists mainly of brushite (dicalcium phosphate dihydrate or CaHPO₄^H₂O). The cement can be made to set in moulds with the desired final shape of the cement block, as granules, rods, sheets, sponges, or other shapes.

In a second step, the set brushite cement can be pulverised in the form of granules, or its shape adapted to the intended use by fragmentation, abrasion, or filing. The size of the granules may be between 0.05 and 4.0 mm. Preferably the size of the granules is in the range between 0.2 and

2.0 mm. Presentation in the form of granules or porous matrices prevent the acidification of the medium and the material results more biocompatible. Optionally this pulverisation, fragmentation, abrasion or filing process can take place after the third step of thermal treatment described next.

In a third step, the set brushite cement is converted to monetite by means of thermal treatment between 80 y 300⁰C during a period between 5 minutes and 12 hours, and a relative humidity between 0 y 100%.

Preferably, the temperature is selected between 121 and 160 ⁰C and is applied for 20 minutes to 2 hours. Under appropriate conditions, this process allows to obtain sterile monetite matrices. The proportion of calcium to phosphorous of the monetite matrix varies between 1 and 1.7.

Depending on the materials used, the resulting material can have an acid pH. In this case, to neutralise the acid pH, the material can be washed with buffer solutions or distilled water for a period up to 4 days. For example, and with out restriction, the buffer solutions can be phosphate buffers, citrate buffer, Hank^'s medium, and/or any buffer solution with a pH between 6 and 8. This washing process can be carried out before and/or after the thermal treatment.

In a fourth step the monetite matrices can be combined with bioactive agents that favour the bone regeneration process such as for example, growth factors, hormones, polysaccharides, cells, proteins, peptides, antiinfectives, analgesics, antiinflamatory agents, antibiotics, antigens, or any of their combinations thereof.

Incorporation of said agents can be carried out by means of adsorption or immersion in solutions containing the bioactive. The growth factors include, with out restriction, platelet derived growth factor platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMP), transforming growth factor-beta-1 (TGF-β- 1 ), growth hormone (GH), insulin like growth factor-1 (IGF1 ), insulin like growth factor-2 (IGF2), fibroblast growth factor (FGF) or any of their combinations thereof.

The proteins include, with out restriction, collagen, fibronectin, albumin or any of their combinations thereof. Furthermore undefined media can also be incorporated to promote bone regeneration, such as, with out restriction, blood, serum or plasma.

To improve the stability of the bioactives adsorbed to the monetite matrix, stabilising agents such as trehalose, sucrose, raffinose, manitol, albumin or collagen can be added to the solution containing the bioactive. Among the bioactive agents that can be incorporated to the monetite is strontium that, as previously described, can be incorporated to the cement as an additive to control the setting time. The addition of strontium, as any of its salts and in concentrations up to 10%, permits that blocks, granules, rods, sponges or pellets can be employed in the treatment of osteoporosis.

Blocks and granules of monetite resulting from the thermal treatment of synthetic brushite, with different degrees of porosity, and with the optional addition of bioactive agents, are biocompatible, biodegradable and osteoinductive. These materials are an excellent material for the elaboration of alloplastic grafts with application in bone regeneration, periodontal surgery and implant surgery. In particular, the granular formulation of monetite facilitates a controlled resorption of the material resulting in improved bone regeneration both quantitatively and qualitatively.

Therefore, the term "blocks" refers to a three dimensional monetite or brushite matrix of any shape and size. Blocks can be formed in a mould with a predetermined shape and size, or result from the erosion, abrasion or crushing of a three dimensional matrix for obtainment of the desired shape and size.

The cement described in the present invention has a special interest and application in the elaboration of materials for multiple treatments in bone regeneration with application in traumatology surgery, maxillofacial surgery, periodontal surgery, orthognatic surgery, oral surgery, neurosurgery, palatine fissure treatment, periodontal treatment, treatment of dental conducts, treatment of osteoporotic bone, and alveolar regeneration and horizontal alveolar regeneration, or bone regeneration.

Therefore, other aspect of the invention refers to bone regeneration matrices comprising the synthetic monetite matrix as described above and their use in therapy. Preferably, their use in traumatology surgery, maxillofacial surgery, periodontal surgery, orthognatic surgery, oral surgery, neurosurgery, palatine fissure surgery, dental treatments, osteoporotic bone, alveolar regeneration, bone fissures, bone fusions, or vertical and horizontal bone regeneration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and its variations are not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration and are not intended to be limiting of the present invention.

DESCRIPTION OF THE FIGURES

Figure 1. X-ray diffraction spectra of the synthetic matrix demonstrating phase change from brushite (A) to monetite (B) by thermal treatment (y axis: intensity in arbitrary units; x axis: theta 2 angle).

Figure 2. Scanning electron micrographs of the synthetic matrices demonstrating the change in crystalline structure form brushite (A) to monetite (B) after thermal treatment. The white bar indicates 10 μm.

Figure 3. Brushite granules prior thermal treatment (A) and resulting monetite granules alter thermal treatment (B).

Figure 4. Biopsy from a rabbit calvarium following grafting with granules of brushite (A) and monetite (B). The bone defect grafted with monetite granules resulted in increased bone regeneration.

Figure 5. Optic micrograph of bone defect in a rabbit treated with monetite granules. Complete regeneration of the defect, in black remaining monetite granules surrounded by newly formed bone marked with crosses on grey areas.

Figure 6. Inhibition zone resulting from the antimicrobial effect of a monetite matrix with doxicyclin on a lawn of Actinobacillus actinomycetemcomitans.

Figure 7. Inherent nanoporosity of synthetic monetite crystals as measured by mercury porosimeter.

EXAMPLES

The present invention can be illustrated additionally by means of the following 10 examples, which do not pretend to be limiting. EXAMPLE 1 : Osteoinduction with monetite granules produced from brushite cements.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous and 1.4 g of [beta]-tricalcium phosphate were mixed. To the powder 1 ml/g of a 1 M solution of phosphoric acid was added. Once the cement was set, the brushite solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm (See Figure 3). The resulting brushite granules were treated for 30 minutes in the autoclave at 121 ⁰C, 1 bar of pressure and a relative humidity of 100%. This resulted in the transformation of brushite into monetite and a change in its crystalline structure (See Figures 1 and 2). The brushite and monetite granules obtained were applied separately to a bone regeneration model. Cavities 1 cm in diameter in rabbit calvaria were filled with 0,25 g of the bone inducing materials to be evaluated, that is monetite or brushite. After 4 weeks, post-mortem analysis of the animals demonstrated a higher bone density and larger amount of mineral tissue, and increased resorbtion and tissue increase, in the defects that were made to contain the monetite granules (See Figures 4 and 5).

EXAMPLE 2: Monetite granules that incorporate strontium chloride as a regulator of the rheology of the brushite cement from which they are made of.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous; 1.4 g of [beta]-tricalcium phosphate; 0.11 g of strontium chloride; and 0.05 g of sodium pyrophosphate were mixed. To the powder 0.5 ml/g of a 2 M solution of phosphoric acid was added and mixed for 1 minute. Once the cement was set, the brushita solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm (See Figure 3). To induce the conversion from brushite to monetite and sterilize the material, the resulting brushite granules were thermally treated for 20 minutes in the autoclave at 121 ⁰C, 1 bar of pressure and a relative humidity of 100%. The resulting granules were implanted in the calvaria of New Zeeland rabbits were they demonstrated the capacity to regenerate bone. EXAMPLE 3: Preformed porous monetite matrix produced from a brushite cement containing glucose crystals as pore inducing agent.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous; 1.4 g of [beta]-tricalcium phosphate; 0.11 g of strontium chloride; 0.05 g of sodium pyrophosphate; and 15 mg of glucose as a pore inducing agent were mixed. To the powder 0.66 ml/g of a 2 M solution of phosphoric acid was added and mixed for 1 minute. The cement in the form of a paste was introduced in rectangular moulds of about 0.5 cm³. Setting of the cement provided a rectangular brushite block with an estimated porosity of 10% and a pore size of between 200 nm and 500 μm. The brushite matrices were washed in a phosphate buffer pH 7.4 for 2 days to neutralise their acid pH. To induce the conversion from brushite to monetite and sterilize the material, the resulting brushite blocks were thermally treated in dry heat for 2 hours at 160 ⁰C. The resulting granules were implanted in the calvaria of New Zeeland rabbits were they demonstrated the capacity to regenerate bone.

EXAMPLE 4: Monetite granules that incorporate hialuronic acid as a modulator of the rheology of the brushite cement from which they are made of.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous; 1.4 g of [beta]-tricalcium phosphate; 0.11 g of strontium chloride; and 0.05 g of sodium pyrophosphate. To the powder 0.5 ml/g of a 1 M solution of glycolic acid in high molecular weight hyaluronic acid was added and mixed for 1 minute. Once the cement was set, the brushite solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm. To induce the conversion from brushite to monetite and sterilize the material, the resulting brushite granules were thermally treated in dry heat for 2 hours at 160 ⁰C. The granules were subsequently washed with a phosphate buffer pH 7.4 for 4 days to neutralise the acid pH of the granules. The resulting granules were implanted in the calvaria of New Zeeland rabbits were they demonstrated the capacity to regenerate bone. EXAMPLE 5: Preformed porous monetite matrix produced by inclusion of polylactide particles in the brushite cement and their subsequent dissolution.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous; 1.4 g of [beta]-tricalcium phosphate; and 0,2 g of polylactide acid microparticles between 1 and 10 microns were mixed. To the powder 1 ml/g of a 1 M solution of phosphoric acid was added. The cement in the form of a paste was introduced in rectangular moulds of about 0.5 cm³. Once set, the monetite blocks were washed with dimethylformamide for 2 days which resulted in the dissolution of polylactide particles and a porous structure. To induce the conversion from brushite to monetite and sterilize the material, the resulting brushite blocks were thermally treated in dry heat for 2 hours at 160 ⁰C. The resulting granules were implanted in the calvaria of New Zeeland rabbits were they demonstrated the capacity to regenerate bone.

EXAMPLE 6: Monetite granules containing doxicyclin.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous; 1.4 g of [beta]-tricalcium phosphate; and 200 mg of hydroxyapatite were mixed. To the powder 0.5 ml/g of a 1 M solution of phosphoric acid was added and mixed for 1 minute. Once the cement was set, the brushita solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm. To induce the conversion from brushite to monetite and sterilize the material, the resulting brushite granules were thermally treated in a humid environment for 20 minutes at 160 ⁰C and 1 bar of pressure. The resulting monetite granules were soaked in 10% doxycyclin hyclate for the incorporation of the antibiotic to the monetite matrix. The material was subsequently treated with 5mg/ml albumin solution. The resulting granules were placed on Petri dishes containing blood agar and previously inoculated with Actinobacillus actinomycetemcomitans, a bacterium frequently associated with pathogenesis of the oral cavity. The presence of doxycyclin resulted in the inhibition of bacterial colonisation of the granules by A. actinomycetemcomitans (See Figure 6). EXAMPLE 7: Porous monetite granules with fibroblast growth factor.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous; 1.4 g of [beta]-tricalcium phosphate; and 30 mg of glucose as a pore inducing agent, were mixed. To the powder 0.5 ml/g of a 0.5 M solution of citric acid was added and mixed for 1 minute. Once the cement was set, the brushite solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm and an estimated porosity of about 10% and a pore size between 200 and 500 μm. Subsequently granules were washed with a phosphate buffer pH 7.4 for 2 days to neutralise the initial acid pH of the granules. To induce the conversion of brushite into monetite and sterilize the material, the brushite granules were thermally treated for 20 minutes in the autoclave at 121 ⁰C, 1 bar of pressure and a relative humidity of 100%. Part of the material was soaked in a sterile 0.5% collagen solution containing fibroblast growth factor (FGF) and 10% trehalose and was after left to dry by evaporation in a laminar airflow cabinet. Trehalose was added to avoid possible degradation of the FGF during the drying process and subsequent storage at room temperature waiting to perform cell based assays. Granules were added to a tissue culture plate previously seeded with a human fibroblast cell line (MRC-5) in a serum-free medium. After 72 hours fibroblasts were found to preferentially colonise the FGF containing material.

EXAMPLE 8: Preformed porous monetite blocks embedded in collagen and populated by osteoblasts.

For the powder component of a cement, 0.8 g of calcium monophosphate anhydrous and 1.4 g of [beta]-tricalcium phosphate and 30 mg sodium bicarbonate as a pore inducing agent, were mixed. To the powder 0,5 ml/g of a 0,5 M solution of citric acid was added and mixed for 1 minute. Once the cement was set, the resulting brushite solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm. To induce the conversion of brushite into monetite and sterilize the material, the obtained brushite granules were treated in an autoclave at 10% humidity, 1 bar of pressure, 121 ⁰C for 20 minutes. The material was then washed in an aqueous 0.01% collagen solution in Dulbecco's Minimum Essential Medium (DMEM) for 6 hours at 37 ⁰C and then washed with fresh DMEM. The material was then exposed to suspensions of 3*10⁵ rat bone marrow derived mesenchymal stem cells previously enriched for 4 passages in osteogenic-supplemented medium (DMEM + 15% Foetal Calf Serum + 100 nM dexamethasone, 50 ug/mL L-ascorbic acid-2-phosphate). After 5 days of culture in osteogenic-suplemented medium osteoblasts were found to be adhered to the granules. The cell-populated materials were implanted in bone defects in rats were they demonstrated their capacity to regenerate bone.

EXAMPLE 9: Comparative Bone regeneration capabilities of different monetite granules.

Monetite samples have an inherent nanoporosity (broadly defined as 5 - 5,000 nanometre pores) of 30-40% of volume due to the nanometric spaces between the mineral crystals (see Figure 7). However, the presence of larger pores that facilitate cellular colonisation is generally recognised as an important feature in tissue engineering procedures. In the present example material porosity, broadly defined as the space occupied by the larger 5 - 1 ,000 micron pores, was optimized for improved cell in-growth capacity and bone induction. For this, monetite granules with different porosities were manufactured by means of incorporating different amounts of sodium bicarbonate in the powder reaction mixture of cement leading to the formation of brushite. For the powder component of the cement, 0.8 g of calcium monophosphate anhydrous and 1.4 g of beta]-tricalcium phosphate were mixed with different amounts of sodium bicarbonate ranging from 0% to 5% in weight. To the powder 1 ml/g of a 1 M solution of phosphoric acid was added. Once the cements were set, the brushite solids were pulverised and sieved to obtain granular materials with a particle size between 0.2 and 2 mm (See Figure 3). The resulting brushite granules were treated for 30 minutes in the autoclave at 121 ⁰C, 1 bar of pressure and a relative humidity of 100%, for their conversion to monetite. Porosity was evaluated by means of image analysis of micrographs. Total porosity, induced porosity plus the inherent material nanoporosity, was evaluated by means of mercury porosimeter. Resulting monetite granules with different porosities were evaluated for their capability to support cell growth and bone formation capability.

In vitro evaluation of cell growth was carried out by in vitro culture of rabbit bone marrow stromal stem cells in the presence of the monetite granules. Briefly, rabbit femur bone marrow was harvested and suspended in 10ml of minimum essential medium with Earle's salt, and glutamine and nonessential amino acid (E-MEM). The medium was supplemented with 1 mM sodium pyruvate, 1.5 g/L sodium bicarbonate, 60 mg/mL kanamycin sulfate and 15% fetal calf serum. Bone marrow suspensions were aliquoted and to them 0.5 g of the granular material was added. Cell growth was determined through crystal violet staining after 1 and 3 days incubation and MTT assay (according to standard method from Mosman J. Immunol. Meth. 65: 55-63, 1983). In addition, alkaline phosphatase activity was determined as an indicator of osteoblastic differentiation.

In vivo bone formation capability was determined following implantation in the calvaria of New Zeeland rabbits in which a 1 cm diameter hole had been perforated. Evaluation of new bone formation was carried out 6 weeks after implantation of the test material.

EXAMPLE 10: Bone regeneration capabilities of monetite granules tested in human patients.

Monetite granules were produced by mixing 0.8 g of calcium monophosphate anhydrous and 1.4 g of [beta]-tricalcium phosphate and to the mixture 1 ml/g of a 1 M solution of phosphoric acid was added. Once the cement was set, the brushite solid was pulverised and sieved to obtain a particle size between 0.2 and 2 mm. The resulting brushite granules were treated for 30 minutes in the autoclave at 121 ⁰C, 1 bar of pressure and a relative humidity of 100%, resulting in their conversion to monetite.

Upon approval by the Ethical Committee of the Hospital San Carlos (Madrid, Spain) five patients with periodontal bone loss and requiring new tooth implantation were treated in a split-face design with monetite granules on one side of the inferior jaw and commercially available bovine bone regeneration matrix on the other. Briefly, patients between 45 and 70 years of age were admitted to ambulatory surgery and inferior anterior teeth were surgically removed. The commercial bovine granules and monetite test granules were separately mixed with the patient's blood obtained from the point of tooth extraction and each applied to the alveolar sockets in a split mouth design. The gum was sutured with 3o silk and patients requested for a 1 week, follow by a monthly follow up for adverse reactions. After 3 months metal implants were implanted following standard periodontal surgical procedures. Briefly, the gum of patients was cut open to permit the implantation of metal screw-on fixtures. Locations treated with monetite showed a bone-like appearance while locations treated with the commercial bovine hydroxyapatite retained a granular structure similar to the day it was implanted. The drill used to perforate the newly formed bone for subsequent implantation was hollow to permit the extraction of a biopsy for subsequent analysis. Metal implants were inserted at the drill points and the gum was re-sutured. Histomorphometric analysis of the biopsies following their preparation and staining with basic fuchsine and toluidine blue revealed that in alveoli implanted with monetite granules new bone represented 70% while alveoli implanted with commercial bovine hydroxyapatite granules new bone only represented 50%.

Claims

1. A synthetic monetite matrix (CaHPO₄) characterised by having a crystal size of between 0.5 and 25 microns.

2. The synthetic monetite matrix according to claim 1 which is presented in the form of granules between 0.2 and 2 mm in diameter.

3. The synthetic monetite matrix according to claim 1 which is presented in the form of blocks.

4. The synthetic monetite matrix according to any of claims 1 to 3 with an induced porosity, between 0 and 20%.

5. The synthetic monetite matrix according to claims 1 to 4 with a total porosity between 35-60%.

6. The synthetic monetite matrix (CaHPO₄) according to any claims 1 to

5. which is in the form of granules or blocks, and is characterised by having a crystal size of between 0.5 and 25 microns, and total porosity between 35 and 60%.

7. The synthetic monetite matrices according to any of claims 1 to 6 which incorporate at least one bioactive agent.

8. The synthetic monetite matrices according to claim 7 in which at least one of the bioactive agents is selected from hormones, polysaccharides, cells, proteins, peptides, growth factors, antibiotics, analgesics, antiinflamatories, antiinfectives, antigens, or any of their combinations thereof.

9. The synthetic monetite matrices according to claim 8 in which at least one of the bioactive agents is a blood-derived material, or a defined cell growth or differentiation inducing factor selected from platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), bone morphogenic proteins (BMP), transforming growth factor-beta-1 (TGF-β- 1 ), growth hormone (GH), insulin like growth factor-1 (IGF1 ), insulin like growth factor-2 (IGF2), or fibroblast growth factor (FGF), or any of their combinations thereof.

10. The synthetic monetite matrices according to claim 8 in which at least one of the bioactive agents is a protein selected from collagen, fibronectin, albumin, or chitosan, or any of their combinations thereof.

11. Procedure for obtaining synthetic monetite matrices according to any of claims 1 to 10 which incorporates the following steps: a. Synthesis of a brushite (CaHPO₄^HbO) matrix by setting of a cement resulting from mixing an aqueous phase and a solid phase comprising basic calcium salt and an acid calcium salt b. Conversion of the brushite matrix obtained in step (a) into a monetite matrix.

12. The procedure according to claim 11 for obtaining synthetic monetite matrices according to any of claims 7 to 10 that also includes: c. The incorporation to the monetite obtained in step (b) of at least one bioactive agent.

13. The procedure according to any of claims 11 to 12 that also includes introducing a pore forming agent in the cement in step (a).

14. The procedure according to any of claims 11 to 13 which incorporates setting of the cement in a mould.

15. The procedure according to any of claims 10 to 14 which incorporates a change of shape of the brushite matrix obtained in step (a) or the monetite matrix obtained in steps (b) or (c) by abrasion, erosion, crushing or pulverisation.

16. The procedure according to any of claims 11 to15 in which the basic calcium salt is selected from alpha tricalcium phosphate, beta tricalcium phosphate, hydroxyapatite, tetracalcium phosphate, octacalcium phosphate, calcium oxide or any of their combinations thereof.

17. The procedure according to any of claims 11 to15 in which the acid calcium salt is selected from monocalcium phosphate monohydrate, and monocalcium phosphate anhydrous or combinations thereof.

18. The procedure according to any of claims 11 to 17 in which the conversion of brushite into monetite is carried out by heat treatment at temperatures between 80 y 300⁰C.

19. The procedure according to claim 18 in which the heat treatment is between 121 and 16O⁰C and between 20 minutes and 2 hours.

20. The procedure according to any of claims 13 to 19 in which the pore forming agent introduced in the cement is selected from the list calcium carbonate, calcium bicarbonate, sodium bicarbonate, hydrogen peroxide, soluble inorganic salts, sugars, sugar alcohols, or aminoacids, or any of their combinations thereof.

21. The procedure according to any of claims 11 to 20 in which the solid phase includes additives that control the setting time of the cement selected from: sodium pyrophosphate, calcium pyrophosphate , potassium pyrophosphate, strontium chloride, strontium renalate, strontium phosphate, strontium pyrophosphate, sodium acetate, potassium acetate, sodium citrate, potassium citrate, sodium phosphocitrate, potassium phosphocitrate, sodium sulphate, potasium sulphate, calcium sulphate, sodium dihydrogen pyrophosphate magnesium sulphate, sodium byphosphonates, potassium biphosfonates, chondroitin 4-sulphate, chondroitin 6-sulphate, glycolic acid, sodium glycolate, calcium glycolate, barium titanate, barium sulphate, barium fluoride or any of their combinations thereof.

22. The procedure according to any of claims 11 to 21 , in which the aqueous phase includes a solution at a concentration between 0.5 and 3.5 M of any of the following acids phosphoric, glycolic, tartaric, citric, succinic, malic, lactic, chlorhydric and/or sulphuric or any of their combinations thereof.

23. The procedure according to any of claims 11 to 21 , in which the aqueous phase includes an additive to control the rheology of the cement selected from: chondroitin 4-sulphate, chondroitin 6-sulphate, silica gel, silica gel incorporating chondroitin 4-sulphate, silica gel incorporating chondroitin 6-sulphate, strontium chloride, sodium pyrophosphate, calcium pyrophosphate, any pyrophosphate salt, collagen, chitosan, low molecular weight hyaluronic acid, hyaluronic acid, hyaluronates, dextrans, alginate, xantham gum, or methyl celluloses, or any of their combinations thereof.

24. The procedure according to any of claims 11 to 23 in which the aqueous phase incorporates an additive to control cement cohesion selected from chondroitin 4-sulphate, chondroitin 6-sulphate, or silica gel, or any of their combinations thereof.

25. The procedure according to any of claims 11 to 24 in which the acid pH of the resulting brushite or monetite matrices is neutralized by immersion or washing with water or aqueous buffers.

26. Bone regeneration matrices comprising synthetic monetite matrix according to any of claims 1 to 10, or produced by any procedures according to claims 11 to 25.

27. Bone regeneration matrices according to claim 26 for their use in therapy.

28. Bone regeneration matrices according to claim 27 for their use in traumatology surgery, maxillofacial surgery, periodontal surgery, orthognatic surgery, oral surgery, neurosurgery, palatine fissure surgery, dental treatments, osteoporotic bone, alveolar regeneration, bone fissures, bone fusions, or vertical and horizontal bone regeneration.