WO2010116321A2 - Biopolymer-containing calcium phosphate foam, process for obtaining thereof and use for bone regeneration - Google Patents
Biopolymer-containing calcium phosphate foam, process for obtaining thereof and use for bone regeneration Download PDFInfo
- Publication number
- WO2010116321A2 WO2010116321A2 PCT/IB2010/051475 IB2010051475W WO2010116321A2 WO 2010116321 A2 WO2010116321 A2 WO 2010116321A2 IB 2010051475 W IB2010051475 W IB 2010051475W WO 2010116321 A2 WO2010116321 A2 WO 2010116321A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- calcium phosphate
- process according
- foam
- polymer
- weight percent
- Prior art date
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/0047—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L24/0073—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix
- A61L24/0084—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material with a macromolecular matrix containing fillers of phosphorus-containing inorganic compounds, e.g. apatite
Definitions
- the present invention relates to the field of biomaterials for bone regeneration.
- bone cements can be injectable and this can allow the implantation via minimally invasive surgery. Injectability of bone cements can be achieved taking into account some factors such as: additives, mixing conditions (liquid-to-powder ratio) and cement particle size. The presence of polymeric additives is so far the most important factor leading to improved injectability by means of an increased fluidity of the cement paste.
- a perfect apposition is assured between the bone tissue and material, which is achieved with the easy adapting of cement in geometrically complex shapes. This leads to a stable connection between defect and implant, and to a faster healing process.
- calcium phosphate cements are versatile materials, adapting to various clinical applications, due to the fact that modifying their processing parameters can yield cement formulations with various final properties.
- calcium phosphate cements are very biocompatible, osteoconductive and that they can stimulate bone regeneration. Moreover, they can be successfully used as drug release materials, due to the low temperature setting reaction, which allows fast and easy incorporation of the drug into the cement matrix
- the ideal bone replacing cement should not only be osteoconductive or osteoinductive but as well capable of being resorbed with the same rate the bone tissue grows so that it can be gradually replaced by newly formed bone.
- most of the developed calcium phosphate cements are more resorbable than sintered hydroxyapatite ceramics, they still show relatively slow resorption kinetics and in many cases the cement remains stable in the implanted site during years .
- Calcium phosphate cements are degraded by a cell- mediated resorption, osteoclasts being the main cells responsible for this process. They act on the material by degrading layers of it. This process is very slow when the material is fully dense or microporous.
- nano- and micropores are formed, but the absence of interconnected macropores is indeed a limiting factor to achieve a resorption that is tuned with the apposition of new bone and later participation to its physiological remodelling. Indeed, macroporosity is an important requirement, which enhances the efficacy of cell-mediated resorption. If the designed material has interconnected macropores, cells can move freely through it.
- both new bone apposition and cement resorption will occur not only at the material exterior surface but within the pores thus increasing the rate of healing of the bone defect.
- macroporosity enhances angiogenesis, a process necessary to the survival of the newly formed bone.
- the process of tissue growth can also be stimulated by the incorporation of bone morphogenic proteins (BMP) and other growth factors .
- BMP bone morphogenic proteins
- porogenic agents can be either sugar, or sodium bicarbonate, or phosphate salts .
- 5,023,387 propose the incorporation of biodegradable polymer such as polylactic acid, which degrades over short time period in a physiological fluids forming porosity during cement setting.
- U.S. Pat. No. 6, 670, 293 and 6,547,866 Bl to Edwars et al shows a method by mixing a calcium source and a phosphate source with a carbonate source and mixing this powdered component with a liquid component having an acid component. The acid and the carbonate react to form carbon dioxide, which produces interconnected porosity in the self-hardening bone cement.
- Bohner at al . (E. P. Pat. No. 1150722, U.S. Pat. No. 6,642,285) developed porous implants based on calcium phosphate cements and hydrophobic liquid.
- the cement paste is mixed with the hydrophobic liquid obtaining an emulsion.
- a surface active agent is suggested to be used as an additive with the scope of lowering the surface tension and facilitating the formation of emulsion between the cement paste and hydrophobic acid.
- the composition hardens after mixing and results in a macroporous cement, provided the hydrophobic liquid is removed.
- Ginebra et al . (WO2006/030054 Al) suggest a method for obtaining injectable calcium phosphate foams by the addition of synthetic non-ionic surfactants.
- U.S. Pat. No. 4, 530,905 by Freedman proposes the use of cross-linked gelatine foams, which are water-swellable and water- insoluble. These anhydrous foams can be applied as biosupport for isolation and purification of enzymes and other proteins.
- Neumann presents a method for obtaining gelatine-based stabilized foams by passing a stream of air through the composition of gelatine, an anionic surface active agent and a water soluble ferrous salt.
- gelatine-based foams can be used in the treatment of human burn wounds, where previously bacteria and other drugs can be incorporated into the foam solution and thus, will enhance their medical usefulness.
- soy protein has been shown to possess good foaming properties and, for this reason, it is used as foaming agents in food industry. Moreover, their positive effect in the treatment of various diseases is well documented.
- An invention by Santin et al . 2001 (PCT/GBOl/03464) has proposed the preparation of biomaterials from soy curd by thermosetting.
- an alternative method has been proposed by Santin et al 2008 (PCT/GB2008/051117) where soy-derived biopolymers with controllable composition are obtained by an extraction method from defatted soybean flour.
- the invention also claims the use of these soybean-based biomaterials as biosurfactant .
- the osteogenic potential of soybean-based biomaterials can be attributed to their natural isoflavone content.
- Isoflavones such as the genistein and the daidzein have an estrogenic action on bone and stem cells and can be used for preventing bone loss related to the insurgence of osteoporosis in menopausal women.
- sodium alginate has found various applications in food and pharmaceutical industries due to its lack of toxicity and good solubility. Over the last years, sodium alginate has drawn the attention for distinct medical applications such as for controlled drug release, as promoter of tissue invasion and bone formation in ceramic matrix (Dong Z et al . 2006, Teng S et al . 2006) .
- the present invention deals with a new self setting composite foams based on the mixture of biopolymer-based foams with calcium phosphate cement.
- the invention has achieved foamed cements that can be injected while retaining their interconnected porous structure.
- the resulting macroporous cements are able to harden in physiological conditions thus providing a solid interconnected macroporous network where bone in-growth is promoted by the combined action of the biopolymer bioactivity (e.g. soy isoflavone action on bone cells) and calcium phosphate cement osteoconductivity .
- the biopolymers can be either gelatine, sodium alginate or soybean, or different combinations thereof.
- the advantage of the process of this invention is that foamed materials can be prepared quickly and efficiently under conditions, which neither require special environment, i.e. high temperature, vacuum, etc., nor a special equipment.
- Another object of the present invention relates to self-setting, injectable composite calcium phosphate foams obtainable by the process mentioned on the first object of the invention.
- the resulting cement foams are very stable, have interconnected macropores and can be easily injected.
- Still another object of the present invention relates to the use of composite calcium phosphate foams according to the previous object of the invention as biomaterial in bone regeneration and/or as scaffold for bone tissue engineering .
- Still another object of the present invention relates to the use of composite phosphate foams with soybean and gelatine as polymers for sustaining the controlled release of soy isoflavones in bony tissues.
- Figure 1 SEM image of a composite calcium phosphate foam obtained by mixing a gelatine-soybean polymer foam with a calcium phosphate cement.
- Figure 4 Effect of the biopolymers on the injectability of the calcium phosphate cement paste.
- FIG. 1 Isoflavone release from calcium phosphate discs containing soybean-based hydrogel at 5%, 10% and 20%.
- A Release profile of the main glycosylated isoflavones
- B Release profile of the non-glcyosylated forms.
- Soy gel% indicates the concentration by weight of soybean-based hydrogel in the calcium phosphate discs.
- G indicates the presence of gelatine in the composite.
- the present invention relates to a process for obtaining a composite calcium phosphate foam comprising the steps of: a) forming a biopolymer foam by agitating or gas blowing a polymer aqueous solution comprising gelatin, sodium alginate, soybean derived polymer or combinations thereof; b) mixing the biopolymer obtained in a) with calcium phosphate cement powder.
- This process suggests using gelatine, sodium alginate and/or soybean derived polymers as foaming agents and/or foam stabilisers and as injectability promoters for a calcium phosphate cement.
- the amount of polymer which is employed relative to the water content, or the ratio between one or more additives in the liquid phase can vary depending upon factors such as foaming ability and foam stability of the liquid phase, good cohesion and injectability of the cement foam and existence of highly interconnected macropores.
- the amount of aqueous liquid which is employed depends upon the molecular weight and the amount of the mentioned polymers employed. Typically, poor foaming occurs if the polymer solution is very dilute or the molecular weight of the polymer is too low. Conversely, the formation of desirable foams is a difficult process to perform if the polymer concentration in the aqueous liquid is very high or the molecular weight of the polymer is too high.
- the foaming process is highly dependant upon the rate of agitation to which the aqueous solution is subjected. Little or no foaming occurs when a conventional magnetic stirrer is employed as an agitation device. For this reason, it is necessary to provide a sufficiently high rate of agitation using a device such as a homogenizer, blender, etc.
- This high agitation rate is preferably constant and exceeds 500 rpm, preferably around 1500 rpm and more preferably about 3000 rpm. It is understood that practically any device capable of producing such a high constant agitation rate can be effectively employed.
- the biopolymer solution can be subjected to a high rate of agitation for a variable period of time.
- the period of time over which the solution is subjected to agitation can depend upon factors such as the amount of reactants, the concentration of reactants in the liquid phase, etc.
- the period of time over which the solution is subjected to agitation can range from about few seconds to several minutes.
- step a) of the process described previously a synthetic surfactant can be added in step a) before agitation or gas blowing in order to increase foamability.
- said synthetic surfactant is a non-ionic surfactant, preferably Pluronic, Span or Tween 80, and more preferably said non-ionic surfactant is Tween ⁇ O.
- concentration of Tween ⁇ O in the solution, when used, is lower than 10 wt%, preferably lower than 1 wt%, more preferably 0.5 wt%.
- the solid phase of calcium phosphate cement comprises calcium and phosphorous sources, which upon setting give a product with different final composition, such as dicalcium phosphate dihydrate, octacalcium phosphate, amorphous calcium phosphate, calcium-deficient hydroxyapatite, stoichiometric hydroxyapatite, or carbonated apatite.
- the main reagents of the solid phase can be present as one component or as two or more components.
- Some of the interesting sources for calcium and phosphorous include: tetracalcium phosphate, dicalcium phosphate anhydrous, dicalcium phosphate dihydrate, alpha- tricalcium phosphate, beta-tricalcium phosphate, monocalcium phosphate monohydrate, amorphous calcium phosphate.
- Other interesting components as calcium sources are for example: calcium carbonate, calcium sulphate, calcium oxide or calcium hydroxide. From the sources of phosphorous, all the soluble phosphates and the phosphoric acid can be utilized. After mixing with a liquid phase the raw materials should set.
- the setting reaction depends on the precise selection of the calcium and phosphorous sources which form part of the powder phase, their proportions and characteristics, as it is shown in various patents (Brown and Chow U.S. Pat. No. RE 33,161; U.S. Pat. No. RE 33,221; Chow and Takagi U.S. Pat. No. 5,525,148; Constantz U.S. Pat. No. 4,880,610; U.S. Pat. No .5, 820, 632 ; U.S. Pat. No. 6, 375, 935) .
- the liquid phase can be pure water or aqueous solution of other phosphate salt, which is previously dissolved.
- a water-soluble polymer with various concentrations can be added to the liquid phase of the cement with the aim of improving the cement workability, injectability and shorten the setting times.
- the term "liquid phase” means a liquid comprising water, which can contain soluble additives.
- the traditional recipe for obtaining calcium phosphate cement is mixing a powder form comprising a calcium and phosphorous source with liquid medium. Some accelerating or retarding agents can be used. In some cases it is difficult to extrude the mixture through a thin needle due to press filtering which leads to phase separation. This phase separation is an obstacle to achieve easily injectable cement pastes. Injectable bone cements can be obtained by increasing the liquid-to-powder ratio (L/P) , but this has a negative effect on the mechanical stability due to increased microporosity of the hardened final product. With the intention of improving the rheological properties, the influence of several additives to the liquid phase (e.g.
- a foam consists of gas bubbles whose walls are thin liquid films.
- Foams are thermodynamically unstable since their collapse is accompanied by a decrease in the total free energy.
- the addition of a polymer may extend the life-time of such foams by several hours (U.S. Pat. No. 4,086,331, Schramm 2005)
- foams remain in liquid form, their life-time is depending on the following: a. water evaporation from the liquid surface to the air b. liquid drainage from the bubble walls c. foam aging or maturation - usually related to the different stability of small versus large bubbles due to Ostwald ripening.
- the foam life-time is governed by the rate at which water drains or evaporates from the walls of the bubbles.
- the addition of water-soluble polymer is beneficial in increasing the viscosity of the foam solution and the water holding capacity of the foam hence, decreasing the drainage.
- additives selected from the group consisting of setting accelerants or retardants, nucleating agents, glycerol, lactic acid, citric acid, chitosan, polysaccharides and soluble polymers, are previously added to the calcium phosphate cement powder.
- a nucleating agent is precipitated hydroxyapatite .
- Precipitated hydroxyapatite facilitates the nucleation of the final phase, which precipitates in the cement.
- the amount is usually inferior to 10 wt % and preferably inferior to 5 wt % with regard to the solid phase.
- growth factors and/or drugs are added at any of steps a) or b) .
- the process of mixing foam with cement powder should not exceed several seconds, typically takes about 20 seconds in order not to break the created bubbles. Thereafter, the foamed cement paste can either be mixed with spatula, or can be placed in a chirurgical syringe and injected.
- the injectability of the created cement foam is very high and for all foam mixtures it reaches almost 100% injectability.
- the foaming agents used are natural polymers with excellent foaming ability responsible for creation of highly interconnected macroporous network; on the other side the mixing with calcium sources and phosphorous sources provides certain stability of the foam by increase of the viscosity.
- the foamed calcium phosphate pastes obtained are stable, injectable and are able to set in a physiological environment.
- the foams thus created give highly porous structures with interconnected macropores, together with micro and nanopores.
- calcium ions can also act as cross-linking agents for various hydrogels. It is anticipated therefore, that cement biopolymer-based foams will have an improved degradation rate in vivo due to the elevated porosity, and given their bioactivity the so developed cement foams should have superior bone regeneration potential.
- the present invention also relates to a composite calcium phosphate foam obtainable by the process disclosed in the present invention.
- the present invention also relates to the use of the composite calcium phosphate foam obtainable by the process disclosed in the present invention as a biomaterial in bone regeneration and/or scaffold for bone tissue engineering.
- said scaffold can be shaped or injected with various geometries and set in physiological conditions, both in vitro and in vivo.
- Foaming should be performed at a temperature between about 45 0 C and about 6O 0 C, preferably between about 5O 0 C and about 6O 0 C, which is the temperature of the water bath where the polymer solution is kept during the foaming process.
- gelatine solutions undergo gelation at temperatures below 30 0 C, and at room temperature the solution fluidity will depend upon the polymer concentration.
- gelatine solutions with concentrations between 1 and 40 weight percent, preferably between 1 and 30 weight percent, more preferably between 1 and 20 weight percent should be kept in a warm water bath prior to the foaming process, and the foaming process normally should be performed while the polymer solution is kept inside the water bath.
- An example of a gelatine-based calcium phosphate foam preparation is reported below.
- a solution containing 15 wt% of type B gelatine is prepared using distilled water and 2.5 wt% Na2HPO 4 used as accelerant of the setting reaction.
- Two millilitres of the gelatine solution previously placed in a warm water bath is subjected to foaming for about 1 minute with mini- mixer.
- To this mixture 2.7 g of calcium phosphate cement is added and gently mixed with the created foam.
- the calcium phosphate powder consists of alpha-tricalcium phosphate previously mixed and homogenized with 2 wt% of precipitated hydroxyapatite acting as nucleating agent for the precipitation of hydroxyapatite.
- the cement foam is placed into a syringe with a 2-mm diameter opening and extruded into moulds with various geometries and allowed to set while immersed in Ringer' s solution (aqueous solution containing 0.9 wt% NaCl) at 37 0 C.
- Ringer' s solution aqueous solution containing 0.9 wt% NaCl
- the foamed cement hardens, giving rise to a calcium deficient hydroxyapatite solid foam, with an apparent density of 1.02 g/cm 3 , a skeletal density of 2.47 g/cm 3 and a porosity of 59.1%.
- These foamed cement formulations are also able to harden in blood at 37 0 C.
- soy derived polymer solutions upon agitation give good foams, their stability is rather poor. Therefore, the stability of soy foams is significantly increased when soy solution is added as a second polymer into gelatine solutions.
- the amount of soy extract, prepared as described in PCT/GB2008/051117, in the solution can be from about 10 to 50 weight percent, preferably lower than 30 weight percent, more preferably about 20 weight percent while the gelatine concentration ranges between 1 and 30 weight percent.
- An example of a soybean-containing calcium phosphate foam is given below.
- a solution containing 10wt% of gelatine type B is prepared using distilled water and 2.5 wt% Na2HPO 4 used as accelerant of the setting reaction. Separately, another soy-containing solution with 40 wt% soy and 2.5 wt% Na2HPO 4 is prepared.
- One millilitre of the gelatine solution previously placed in a warm water bath is mixed with 1 ml of the soy extract solution and this mixture is subjected to foaming at a temperature between about 45 0 C and about 6O 0 C, preferably between about 5O 0 C and about 6O 0 C, for about 1 minute with a mini-mixer. To this mixture 3.6 g of calcium phosphate cement is added and gently mixed with the created foam.
- the cement foam is placed into a syringe with a 2-mm diameter opening and extruded into moulds with various geometries and allowed to set while immersed in Ringer' s solution (aqueous solution containing 0.9 wt% NaCl) at 37 0 C.
- Ringer' s solution aqueous solution containing 0.9 wt% NaCl
- the foamed cement hardens, giving rise to a calcium deficient hydroxyapatite solid foam, with an apparent density of 0.88 g/cm 3 , a skeletal density of 2.44 g/cm 3 and a porosity of 63.8%.
- These foamed cement formulations are also able to harden in blood at 37 0 C.
- Sodium alginate is a water soluble natural polysaccharide. When a sodium alginate solution is foamed a poor foamability is achieved, which depends on the molecular weight of the polymer. Besides, alginate act as a cohesion promoter when added to calcium phosphate cements, due to the natural cross-linking in the presence of divalent ions such as calcium in the aqueous media. Normally the contribution of sodium alginate as foaming agent is very low, but on other side sodium alginate acts as good foam stabilizer. In order to improve the foaming ability of alginate solutions, a synthetic surfactant can be used, such as Tween 80, Pluronic or Span.
- Tween 80 was added to the alginate solution with concentrations from about 1 weight percent to about 10 weight percent. Tween concentrations can vary from about 0.01 to 10 wt%.
- An example of alginate-tween-containing calcium phosphate foam is given below.
- a solution containing 2 wt% sodium alginate is prepared using distilled water containing 2.5 wt% Na2HPO4 used as accelerant of the setting reaction, and 0.5 wt% of Tween 80 used as surfactant. Two millilitres of this solution is subjected to foaming at room temperature for about 1 minute with a mini-mixer. To this mixture 3.6 g of calcium phosphate cement is added and gently mixed with the created foam. Thereafter, the cement foam is placed into a syringe with a 2 mm diameter opening and extruded into moulds with various geometries and allowed to set while immersed in Ringer's solution (aqueous solution containing 0.9 wt% NaCl) at 37 0 C.
- Ringer's solution aqueous solution containing 0.9 wt% NaCl
- the foamed cement hardens, giving rise to a calcium deficient hydroxyapatite solid foam, with an apparent density of 1.22 g/cm 3 , a skeletal density of 2.58 g/cm 3 and a porosity of 52.7%.
- foamed cement formulations are also able to harden in blood at 37 0 C.
- the injectability of the material was measured by means of an extrusion test, using a mechanical testing machine, at a constant cross-head speed of 15 mm/min up to a maximum force of 100 N as shown in Figure 3.
- Cement composite injectability is calculated as a ratio between the weight of injected cement paste and the weight of initial cement paste.
- the addition of the biopolymers produced an increase of the injectability of the paste, as compared to the inorganic calcium phosphate cement, as shown in Figure 4.
- the content of the four main soy non- glycosylated isoflavones (genistein and daidzein) and glycosylated isoflavones (genistin and daidzin) was determined by a standard HPLC method. Data were transformed by the use of standard curve obtained from the pure standards and expressed as isoflavone micrograms/ml of supernatant. The data of release for the isoflavone glycosylated forms clearly shows that in absence of gelatine the 20%s soy-containing discs release significantly higher concentrations of isoflavones and the released is increasing gradually over 14 days (Figure 5A) . For the discs containing 5% and 10% soy significantly lower levels were released and reached a plateau already at day 3. In presence of 5% gelatine the release of glycosylated form was significantly lower than in cements without gelatine. In particular, it seems that a plateau is reached already at day 1 and a reduction is detected at day 7 and 14.
- the present invention also relates to the use of the composite calcium phosphate foam for sustaining a controlled release of soy isoflavones in bony tissues.
- Ginebra MP et al . Mechanical and rheological improvement of a calcium phosphate cement by the addition of a polymeric drug, J Biomed Mater Res 2001; 57(1) : 113-118,
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Surgery (AREA)
- Composite Materials (AREA)
- Materials Engineering (AREA)
- Engineering & Computer Science (AREA)
- Inorganic Chemistry (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
The present invention relates to a process for obtaining a composite calcium phosphate foam comprising the steps of : a) forming a biopolymer foam by agitating or gas blowing a polymer aqueous solution comprising gelatin, sodium alginate, soybean derived polymer or combinations thereof; and b) mixing the biopolymer foam obtained in a) with calcium phosphate cement powder. The invention also relates to the composite calcium phosphate foam obtainable by the process of the present invention and its use as a biomaterial in bone regeneration and/or scaffold for bone tissue engineering.
Description
BIOPOLYMER-CONTAINING CALCIUM PHOSPHATE FOAM, PROCESS FOR OBTAINING THEREOF AND USE FOR BONE REGENERATION
1. Field of the invention
The present invention relates to the field of biomaterials for bone regeneration.
2. Description of Related Art
The development of calcium phosphate cements begun more than two decades ago, when Brown and Chow had published their invention on dental cements (U.S. Pat. 4,518,430) . The unique idea arose from the combined usage of two calcium phosphate sources, which can form hydroxyapatite by precipitation. This precipitation is preceded by dissolution of the reagents, which occurs at room temperature. Many combinations between various calcium phosphate cements have been developed so far. By modification of the initial composition, liquid medium and additives, the final product can vary in crystallinity, stoiochimetry and thus, reactivity. Brushite forming calcium phosphate cements have also been proposed (U.S. Pat. No. 6,733,582Bl and U.S. Pat. No. 5,997,624)
The application of calcium phosphate cements, especially in the treatment of bone defects is associated with three important advantages in comparison with bulk ceramics of granules. Firstly, bone cements can be injectable and this can allow the implantation via minimally invasive surgery. Injectability of bone cements can be achieved taking into account some factors such as: additives, mixing conditions (liquid-to-powder ratio) and cement particle size. The presence of polymeric additives is so far the most important factor leading to improved injectability by means of an increased fluidity of the
cement paste. Secondly, a perfect apposition is assured between the bone tissue and material, which is achieved with the easy adapting of cement in geometrically complex shapes. This leads to a stable connection between defect and implant, and to a faster healing process. Lastly, calcium phosphate cements are versatile materials, adapting to various clinical applications, due to the fact that modifying their processing parameters can yield cement formulations with various final properties.
Different studies have shown that calcium phosphate cements are very biocompatible, osteoconductive and that they can stimulate bone regeneration. Moreover, they can be successfully used as drug release materials, due to the low temperature setting reaction, which allows fast and easy incorporation of the drug into the cement matrix
(Ginebra et al . , 2006) . Growth factors, antibiotics and other drugs can be introduced in the cement (either in the powder or liquid phase) , which gradually can be released over time.
The ideal bone replacing cement should not only be osteoconductive or osteoinductive but as well capable of being resorbed with the same rate the bone tissue grows so that it can be gradually replaced by newly formed bone. Although most of the developed calcium phosphate cements are more resorbable than sintered hydroxyapatite ceramics, they still show relatively slow resorption kinetics and in many cases the cement remains stable in the implanted site during years .
Calcium phosphate cements are degraded by a cell- mediated resorption, osteoclasts being the main cells responsible for this process. They act on the material by degrading layers of it. This process is very slow when the material is fully dense or microporous. During the setting
reaction of calcium phosphate cements, nano- and micropores are formed, but the absence of interconnected macropores is indeed a limiting factor to achieve a resorption that is tuned with the apposition of new bone and later participation to its physiological remodelling. Indeed, macroporosity is an important requirement, which enhances the efficacy of cell-mediated resorption. If the designed material has interconnected macropores, cells can move freely through it. When the access of bone progenitor cells and osteoclasts to the bulk material is facilitated, both new bone apposition and cement resorption will occur not only at the material exterior surface but within the pores thus increasing the rate of healing of the bone defect. In addition, macroporosity enhances angiogenesis, a process necessary to the survival of the newly formed bone. The process of tissue growth can also be stimulated by the incorporation of bone morphogenic proteins (BMP) and other growth factors .
Various methods for introducing pores in ceramics and cements have been described. Some, such as Chow et al .
(U.S. Pat. No. 5, 525, 148) suggest to incorporate a solid phase insoluble in the cement that can be eliminated by dissolution in physiological fluids. Such porogenic agents can be either sugar, or sodium bicarbonate, or phosphate salts .
Others, such as Hirano et al . (J. P. Pat. No.
5,023,387) propose the incorporation of biodegradable polymer such as polylactic acid, which degrades over short time period in a physiological fluids forming porosity during cement setting.
U.S. Pat. No. 5, 820,632 to Constanz et al . suggests the addition of aggregates of soluble materials, generally above 25 volume per cent which may leach out or produce
gas during the reaction, thus will provide porosity in the cement. Specifically, Constanz et al . propose the use of calcium chloride and sodium or potassium hydroxide, which are water-soluble and will be leached out to form porosity inside the cement.
U.S. Pat. No. 6, 670, 293 and 6,547,866 Bl to Edwars et al . shows a method by mixing a calcium source and a phosphate source with a carbonate source and mixing this powdered component with a liquid component having an acid component. The acid and the carbonate react to form carbon dioxide, which produces interconnected porosity in the self-hardening bone cement.
Bohner at al . (E. P. Pat. No. 1150722, U.S. Pat. No. 6,642,285) developed porous implants based on calcium phosphate cements and hydrophobic liquid. The cement paste is mixed with the hydrophobic liquid obtaining an emulsion. Eventually, a surface active agent is suggested to be used as an additive with the scope of lowering the surface tension and facilitating the formation of emulsion between the cement paste and hydrophobic acid. The composition hardens after mixing and results in a macroporous cement, provided the hydrophobic liquid is removed.
Ginebra et al . (WO2006/030054 Al) suggest a method for obtaining injectable calcium phosphate foams by the addition of synthetic non-ionic surfactants.
U.S. Pat. No. 6,485,754 Bl to Wenz et al . shows a method for incorporation of cationic antibiotic salt to bioresorbable hydroxyapatite-like compounds to obtain an injectable, curable bone cement which acts as a depot for controlled release of the drug over long period of time.
However, all these methods do not ensure an open pore macroporosity of the material upon and following implantation by injection.
With regards to polymer foams, U.S. Pat. No. 4, 530,905 by Freedman proposes the use of cross-linked gelatine foams, which are water-swellable and water- insoluble. These anhydrous foams can be applied as biosupport for isolation and purification of enzymes and other proteins.
Neumann (U.S. Pat. No. 4,086,331) presents a method for obtaining gelatine-based stabilized foams by passing a stream of air through the composition of gelatine, an anionic surface active agent and a water soluble ferrous salt. These gelatine-based foams can be used in the treatment of human burn wounds, where previously bacteria and other drugs can be incorporated into the foam solution and thus, will enhance their medical usefulness.
Other substances that might be interesting for these types of applications are soy components. In particular, soy protein has been shown to possess good foaming properties and, for this reason, it is used as foaming agents in food industry. Moreover, their positive effect in the treatment of various diseases is well documented. An invention by Santin et al . 2001 (PCT/GBOl/03464) has proposed the preparation of biomaterials from soy curd by thermosetting. Recently, an alternative method has been proposed by Santin et al 2008 (PCT/GB2008/051117) where soy-derived biopolymers with controllable composition are obtained by an extraction method from defatted soybean flour. These novel soybean-based biomaterials have been shown to enhance osteoblasts differentiation and bone repair in vivo. Among other claims, the invention also claims the use of these soybean-based biomaterials as
biosurfactant . According to Ishimi et al . 1999 and other papers (e.g. Morris et al 2006), the osteogenic potential of soybean-based biomaterials can be attributed to their natural isoflavone content. Isoflavones such as the genistein and the daidzein have an estrogenic action on bone and stem cells and can be used for preventing bone loss related to the insurgence of osteoporosis in menopausal women.
Sodium alginate has found various applications in food and pharmaceutical industries due to its lack of toxicity and good solubility. Over the last years, sodium alginate has drawn the attention for distinct medical applications such as for controlled drug release, as promoter of tissue invasion and bone formation in ceramic matrix (Dong Z et al . 2006, Teng S et al . 2006) .
The present invention deals with a new self setting composite foams based on the mixture of biopolymer-based foams with calcium phosphate cement. The invention has achieved foamed cements that can be injected while retaining their interconnected porous structure. The resulting macroporous cements are able to harden in physiological conditions thus providing a solid interconnected macroporous network where bone in-growth is promoted by the combined action of the biopolymer bioactivity (e.g. soy isoflavone action on bone cells) and calcium phosphate cement osteoconductivity .
SUMMARY OF THE INVENTION
It is an object of the present invention to describe a process for obtaining composite calcium phosphate foams by combining solutions of biopolymers with foaming properties with calcium phosphate powders; the final mixture having the ability to set through a cementitious
reaction in the biological environment of the body. The biopolymers can be either gelatine, sodium alginate or soybean, or different combinations thereof.
The advantage of the process of this invention is that foamed materials can be prepared quickly and efficiently under conditions, which neither require special environment, i.e. high temperature, vacuum, etc., nor a special equipment.
Another object of the present invention relates to self-setting, injectable composite calcium phosphate foams obtainable by the process mentioned on the first object of the invention. The resulting cement foams are very stable, have interconnected macropores and can be easily injected.
Still another object of the present invention relates to the use of composite calcium phosphate foams according to the previous object of the invention as biomaterial in bone regeneration and/or as scaffold for bone tissue engineering .
Still another object of the present invention relates to the use of composite phosphate foams with soybean and gelatine as polymers for sustaining the controlled release of soy isoflavones in bony tissues.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. SEM image of a composite calcium phosphate foam obtained by mixing a gelatine-soybean polymer foam with a calcium phosphate cement.
Figure 2. The same foam at higher magnification
Figure 3: Experimental set up of the injectability test performed (Example 4)
Figure 4. Effect of the biopolymers on the injectability of the calcium phosphate cement paste. The codes used are
as follows: Control-0.4 : inorganic cement, L/P=O .4ml/g; 2AsA-O.80: 2% sulphated sodium alginate, L/P=0.80 ml/g; 15GeI-O.47: 15% gelatine, L/P=0.47ml/g; 20soy-0.32=20%soy extract, L/P=0.32.
Figure 5. Isoflavone release from calcium phosphate discs containing soybean-based hydrogel at 5%, 10% and 20%. (A) Release profile of the main glycosylated isoflavones, (B) Release profile of the non-glcyosylated forms. Soy gel% indicates the concentration by weight of soybean-based hydrogel in the calcium phosphate discs. G indicates the presence of gelatine in the composite.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to a process for obtaining a composite calcium phosphate foam comprising the steps of: a) forming a biopolymer foam by agitating or gas blowing a polymer aqueous solution comprising gelatin, sodium alginate, soybean derived polymer or combinations thereof; b) mixing the biopolymer obtained in a) with calcium phosphate cement powder.
This process suggests using gelatine, sodium alginate and/or soybean derived polymers as foaming agents and/or foam stabilisers and as injectability promoters for a calcium phosphate cement. The amount of polymer which is employed relative to the water content, or the ratio between one or more additives in the liquid phase can vary depending upon factors such as foaming ability and foam stability of the liquid phase, good cohesion and injectability of the cement foam and existence of highly interconnected macropores. The amount of aqueous liquid which is employed, depends upon the molecular weight and
the amount of the mentioned polymers employed. Typically, poor foaming occurs if the polymer solution is very dilute or the molecular weight of the polymer is too low. Conversely, the formation of desirable foams is a difficult process to perform if the polymer concentration in the aqueous liquid is very high or the molecular weight of the polymer is too high.
The foaming process is highly dependant upon the rate of agitation to which the aqueous solution is subjected. Little or no foaming occurs when a conventional magnetic stirrer is employed as an agitation device. For this reason, it is necessary to provide a sufficiently high rate of agitation using a device such as a homogenizer, blender, etc. This high agitation rate is preferably constant and exceeds 500 rpm, preferably around 1500 rpm and more preferably about 3000 rpm. It is understood that practically any device capable of producing such a high constant agitation rate can be effectively employed. The biopolymer solution can be subjected to a high rate of agitation for a variable period of time. For example, the period of time over which the solution is subjected to agitation can depend upon factors such as the amount of reactants, the concentration of reactants in the liquid phase, etc. Typically, the period of time over which the solution is subjected to agitation can range from about few seconds to several minutes.
Another factor which could have an effect on the foam properties (i.e. foaming ability and stability) is the vessel/container/bowl/pot into which the foaming is carried out. Preferably, the dimensions of the plastic container should not exceed too much the diameter of the homogenizer employed as a foam creator. Another way of achieving optimal agitation conditions is by means of gas blowing .
In a preferred embodiment of the step a) of the process described previously, a synthetic surfactant can be added in step a) before agitation or gas blowing in order to increase foamability.
In a more preferred embodiment, said synthetic surfactant is a non-ionic surfactant, preferably Pluronic, Span or Tween 80, and more preferably said non-ionic surfactant is TweenδO. The concentration of TweenδO in the solution, when used, is lower than 10 wt%, preferably lower than 1 wt%, more preferably 0.5 wt%.
The solid phase of calcium phosphate cement comprises calcium and phosphorous sources, which upon setting give a product with different final composition, such as dicalcium phosphate dihydrate, octacalcium phosphate, amorphous calcium phosphate, calcium-deficient hydroxyapatite, stoichiometric hydroxyapatite, or carbonated apatite. The main reagents of the solid phase can be present as one component or as two or more components. Some of the interesting sources for calcium and phosphorous include: tetracalcium phosphate, dicalcium phosphate anhydrous, dicalcium phosphate dihydrate, alpha- tricalcium phosphate, beta-tricalcium phosphate, monocalcium phosphate monohydrate, amorphous calcium phosphate. Other interesting components as calcium sources are for example: calcium carbonate, calcium sulphate, calcium oxide or calcium hydroxide. From the sources of phosphorous, all the soluble phosphates and the phosphoric acid can be utilized. After mixing with a liquid phase the raw materials should set. The setting reaction depends on the precise selection of the calcium and phosphorous sources which form part of the powder phase, their proportions and characteristics, as it is shown in various patents (Brown and Chow U.S. Pat. No. RE 33,161; U.S. Pat.
No. RE 33,221; Chow and Takagi U.S. Pat. No. 5,525,148; Constantz U.S. Pat. No. 4,880,610; U.S. Pat. No .5, 820, 632 ; U.S. Pat. No. 6, 375, 935) .
The liquid phase can be pure water or aqueous solution of other phosphate salt, which is previously dissolved. As already mentioned a water-soluble polymer with various concentrations can be added to the liquid phase of the cement with the aim of improving the cement workability, injectability and shorten the setting times. As used herein, the term "liquid phase" means a liquid comprising water, which can contain soluble additives.
The traditional recipe for obtaining calcium phosphate cement is mixing a powder form comprising a calcium and phosphorous source with liquid medium. Some accelerating or retarding agents can be used. In some cases it is difficult to extrude the mixture through a thin needle due to press filtering which leads to phase separation. This phase separation is an obstacle to achieve easily injectable cement pastes. Injectable bone cements can be obtained by increasing the liquid-to-powder ratio (L/P) , but this has a negative effect on the mechanical stability due to increased microporosity of the hardened final product. With the intention of improving the rheological properties, the influence of several additives to the liquid phase (e.g. glycerol, lactic acid, citric acid, chitosan, polysaccharides or soluble polymers) on the injectability of calcium phosphate bone cements has been studied by various authors (Ginebra MP et al. 2001, Khairoun et al . 1998, Andrianj otovo H et al . 1995, Leroux L et al . 1999, WO2004/103419 Al, U.S. Pat. No. 2004/0244651 Al, U.S. Pat. No. 2005/0199156 Al) . Based on the results obtained by these authors, it can be concluded that the factors controlling injectability of calcium phosphate cements are: increase on L/P ratio, use
of round starting particles, addition of citrate ions and viscous polymer solutions. A different and interesting approach to increase the injectability of a cement paste is to foam it. In other words - increase the macroporosity of cement paste using various ways for foam forming.
As it is known, a foam consists of gas bubbles whose walls are thin liquid films. Usually pure liquids do not foam, and short-live aqueous foams can be generated by passing a stream of air though a solution containing a surface active agent. Foams are thermodynamically unstable since their collapse is accompanied by a decrease in the total free energy. The addition of a polymer may extend the life-time of such foams by several hours (U.S. Pat. No. 4,086,331, Schramm 2005) However, since such foams remain in liquid form, their life-time is depending on the following: a. water evaporation from the liquid surface to the air b. liquid drainage from the bubble walls c. foam aging or maturation - usually related to the different stability of small versus large bubbles due to Ostwald ripening.
These factors lead to gradual thinning and weakening of the bubble wall until is no longer thermodynamically stable and collapses, i.e. the foam life-time is governed by the rate at which water drains or evaporates from the walls of the bubbles. The addition of water-soluble polymer is beneficial in increasing the viscosity of the foam solution and the water holding capacity of the foam hence, decreasing the drainage.
Accordingly, as stated above, in another preferred embodiment of this invention, additives selected from the group consisting of setting accelerants or retardants, nucleating agents, glycerol, lactic acid, citric acid,
chitosan, polysaccharides and soluble polymers, are previously added to the calcium phosphate cement powder.
An non-limiting example of a nucleating agent is precipitated hydroxyapatite . Precipitated hydroxyapatite facilitates the nucleation of the final phase, which precipitates in the cement. The amount is usually inferior to 10 wt % and preferably inferior to 5 wt % with regard to the solid phase.
In another preferred embodiment of this invention, growth factors and/or drugs are added at any of steps a) or b) .
The process of mixing foam with cement powder should not exceed several seconds, typically takes about 20 seconds in order not to break the created bubbles. Thereafter, the foamed cement paste can either be mixed with spatula, or can be placed in a chirurgical syringe and injected. The injectability of the created cement foam is very high and for all foam mixtures it reaches almost 100% injectability.
To produce injectable and porous calcium phosphate cements based on alginate, a modification of this biopolymer was also achieved to obtain sulphated alginate.
The advantages of creating such foams are twofold: on one side the foaming agents used are natural polymers with excellent foaming ability responsible for creation of highly interconnected macroporous network; on the other side the mixing with calcium sources and phosphorous sources provides certain stability of the foam by increase of the viscosity. The foamed calcium phosphate pastes obtained are stable, injectable and are able to set in a physiological environment. The foams thus created give
highly porous structures with interconnected macropores, together with micro and nanopores. Moreover, calcium ions can also act as cross-linking agents for various hydrogels. It is anticipated therefore, that cement biopolymer-based foams will have an improved degradation rate in vivo due to the elevated porosity, and given their bioactivity the so developed cement foams should have superior bone regeneration potential.
The present invention also relates to a composite calcium phosphate foam obtainable by the process disclosed in the present invention.
Due to the characteristics of this new material, the present invention also relates to the use of the composite calcium phosphate foam obtainable by the process disclosed in the present invention as a biomaterial in bone regeneration and/or scaffold for bone tissue engineering.
Preferably, said scaffold can be shaped or injected with various geometries and set in physiological conditions, both in vitro and in vivo.
The following examples are included to further illustrate the scope of this invention.
EXAMPLE 1
When gelatine is used alone as foaming agent, the temperature at which the process is performed is very important. Foaming should be performed at a temperature between about 450C and about 6O0C, preferably between about 5O0C and about 6O0C, which is the temperature of the water bath where the polymer solution is kept during the foaming process. As is known, gelatine solutions undergo gelation at temperatures below 300C, and at room temperature the solution fluidity will depend upon the polymer concentration. Preferably, gelatine solutions with
concentrations between 1 and 40 weight percent, preferably between 1 and 30 weight percent, more preferably between 1 and 20 weight percent, should be kept in a warm water bath prior to the foaming process, and the foaming process normally should be performed while the polymer solution is kept inside the water bath. An example of a gelatine-based calcium phosphate foam preparation is reported below.
A solution containing 15 wt% of type B gelatine is prepared using distilled water and 2.5 wt% Na2HPO4 used as accelerant of the setting reaction. Two millilitres of the gelatine solution previously placed in a warm water bath is subjected to foaming for about 1 minute with mini- mixer. To this mixture 2.7 g of calcium phosphate cement is added and gently mixed with the created foam. The calcium phosphate powder consists of alpha-tricalcium phosphate previously mixed and homogenized with 2 wt% of precipitated hydroxyapatite acting as nucleating agent for the precipitation of hydroxyapatite. Thereafter, the cement foam is placed into a syringe with a 2-mm diameter opening and extruded into moulds with various geometries and allowed to set while immersed in Ringer' s solution (aqueous solution containing 0.9 wt% NaCl) at 37 0C. In these conditions, the foamed cement hardens, giving rise to a calcium deficient hydroxyapatite solid foam, with an apparent density of 1.02 g/cm3, a skeletal density of 2.47 g/cm3 and a porosity of 59.1%. These foamed cement formulations are also able to harden in blood at 37 0C.
EXAMPLE 2
Although soy derived polymer solutions upon agitation give good foams, their stability is rather poor. Therefore, the stability of soy foams is significantly increased when soy solution is added as a second polymer into gelatine solutions. The amount of soy extract, prepared as described in PCT/GB2008/051117, in the solution can be
from about 10 to 50 weight percent, preferably lower than 30 weight percent, more preferably about 20 weight percent while the gelatine concentration ranges between 1 and 30 weight percent. An example of a soybean-containing calcium phosphate foam is given below.
A solution containing 10wt% of gelatine type B is prepared using distilled water and 2.5 wt% Na2HPO4 used as accelerant of the setting reaction. Separately, another soy-containing solution with 40 wt% soy and 2.5 wt% Na2HPO4 is prepared. One millilitre of the gelatine solution previously placed in a warm water bath is mixed with 1 ml of the soy extract solution and this mixture is subjected to foaming at a temperature between about 450C and about 6O0C, preferably between about 5O0C and about 6O0C, for about 1 minute with a mini-mixer. To this mixture 3.6 g of calcium phosphate cement is added and gently mixed with the created foam. Thereafter, the cement foam is placed into a syringe with a 2-mm diameter opening and extruded into moulds with various geometries and allowed to set while immersed in Ringer' s solution (aqueous solution containing 0.9 wt% NaCl) at 37 0C. In these conditions, the foamed cement hardens, giving rise to a calcium deficient hydroxyapatite solid foam, with an apparent density of 0.88 g/cm3, a skeletal density of 2.44 g/cm3 and a porosity of 63.8%. These foamed cement formulations are also able to harden in blood at 37 0C.
EXAMPLE 3
Sodium alginate is a water soluble natural polysaccharide. When a sodium alginate solution is foamed a poor foamability is achieved, which depends on the molecular weight of the polymer. Besides, alginate act as a cohesion promoter when added to calcium phosphate cements, due to the natural cross-linking in the presence of divalent ions
such as calcium in the aqueous media. Normally the contribution of sodium alginate as foaming agent is very low, but on other side sodium alginate acts as good foam stabilizer. In order to improve the foaming ability of alginate solutions, a synthetic surfactant can be used, such as Tween 80, Pluronic or Span. Preferably, to the alginate solution with concentrations from about 1 weight percent to about 10 weight percent, Tween 80 was added. Tween concentrations can vary from about 0.01 to 10 wt%. An example of alginate-tween-containing calcium phosphate foam is given below.
A solution containing 2 wt% sodium alginate is prepared using distilled water containing 2.5 wt% Na2HPO4 used as accelerant of the setting reaction, and 0.5 wt% of Tween 80 used as surfactant. Two millilitres of this solution is subjected to foaming at room temperature for about 1 minute with a mini-mixer. To this mixture 3.6 g of calcium phosphate cement is added and gently mixed with the created foam. Thereafter, the cement foam is placed into a syringe with a 2 mm diameter opening and extruded into moulds with various geometries and allowed to set while immersed in Ringer's solution (aqueous solution containing 0.9 wt% NaCl) at 37 0C. In these conditions, the foamed cement hardens, giving rise to a calcium deficient hydroxyapatite solid foam, with an apparent density of 1.22 g/cm3, a skeletal density of 2.58 g/cm3 and a porosity of 52.7%. These foamed cement formulations are also able to harden in blood at 37 0C.
EXAMPLE 4
An extrusion test was performed using a commercial syringe, in order to assess the effect of the biopolymers on the injectability of the cement paste. This test was carried out in non-foamed pastes. Three biopolymer solutions were used: 15% gelatine, 2% sodium alginate and
20% soy extract, and were mixed with the calcium phosphate powder. An inorganic calcium phosphate cement was used as a control. The liquid to powder ratio was adjusted in each formulation to obtain a workable paste. The resulting pastes were placed into commercial syringes with an opening of 0.18 mm. The injectability of the material was measured by means of an extrusion test, using a mechanical testing machine, at a constant cross-head speed of 15 mm/min up to a maximum force of 100 N as shown in Figure 3. Cement composite injectability is calculated as a ratio between the weight of injected cement paste and the weight of initial cement paste. The addition of the biopolymers produced an increase of the injectability of the paste, as compared to the inorganic calcium phosphate cement, as shown in Figure 4.
EXAMPLE 5
An isoflavone delivery test was performed in those calcium phosphate formulations including the soy extract. Calcium phosphate cements were allowed to set in form of discs without and with 5% (w/w) gelatine and always enriched by different concentrations of soybean-based hydrogels (5%, 10% and 20% by weight) . The discs were incubated in 1 ml of phosphate buffered saline pH 7.2, 37 0C, static conditions at day 1, 3, 7, 14 days. As the materials were set in Ringer' s solution, the release of isoflavones in this experiments does not take into account a possible released occurred during the cement setting. Supernatants from the release experiments were withdrawn and stored at -70 0C until use. The content of the four main soy non- glycosylated isoflavones (genistein and daidzein) and glycosylated isoflavones (genistin and daidzin) was determined by a standard HPLC method. Data were transformed by the use of standard curve obtained from the pure standards and expressed as isoflavone micrograms/ml of supernatant. The data of release for the isoflavone
glycosylated forms clearly shows that in absence of gelatine the 20%s soy-containing discs release significantly higher concentrations of isoflavones and the released is increasing gradually over 14 days (Figure 5A) . For the discs containing 5% and 10% soy significantly lower levels were released and reached a plateau already at day 3. In presence of 5% gelatine the release of glycosylated form was significantly lower than in cements without gelatine. In particular, it seems that a plateau is reached already at day 1 and a reduction is detected at day 7 and 14.
The study of the released of the biologically active, non- glycosylated forms which have lower water solubility showed a gradual increase of the released levels of both genistein and daidzein over 14 days (Figure 5B) . In particular, it is evident that gelatine was favouring the release of these relatively hydrophobic molecules to reach a plateau at day 7. When the data of release of genistein from discs containing 5% gelatine and 10% and 20% soy were converted into genistein concentration (micromoles/litre) , it was observed that the levels ranged from 1.0 to 15 micromoles/litre, a range of concentrations which has been previously proven to be effective on osteoblast differentiation in vitro.
Overall, the experiments have demonstrated that, even when setting of the soy-containing cements is performed in vitro in Ringer' s solution, a significant release of isoflavones takes place over at least 7 days. In particular, the bioactive non-glycosylated form release seems to be improved by the presence of gelatine. The data of these experiments demonstrate that the chosen formulation of cement containing 5% gelatine and 20% soy can sustain the release of isoflavones within a range of time fundamental to the early phase of bone repair and are
therefore likely to accelerate the early deposition of new tissue .
Accordingly, since these experimental conditions reproduced those encountered in bony tissues, it can be stated that the present invention also relates to the use of the composite calcium phosphate foam for sustaining a controlled release of soy isoflavones in bony tissues.
References Cited: U.S. PATENT DOCUMENTS
U.S. Pat. No. 4,518,430
U.S. Pat. No. 6,733,582Bl
U.S. Pat. No. 5, 997, 624
U.S. Pat. No. 5, 525,148
U.S. Pat. No. 5, 820,632
U.S. Pat. No. 6, 670, 293
U.S. Pat. No. 6,547, 866 Bl
U.S. Pat. No. 6, 642,285
U.S. Pat. No. 4, 530,905
U.S. Pat. No. 6,485,754 Bl
U.S. Pat. No. 2004/0244651 Al
U.S. Pat. No. 2005/0199156 Al
U.S. Pat. No. 4, 086,331
U.S. Pat. No. 4,530, 905
U.S. Pat. No. RE 33,161;
U.S. Pat. No. RE 33,221;
U.S. Pat. No. 4, 880, 610;
U.S. Pat. No. 6,375,935
OTHER PATENT DOCUMENTS
J. P. Pat. No. 5,023,387 E. P. Pat. No. 1150722 WO2006/030054 Al PCT/GBOl/03464 PCT/GB2008/051117
OTHER PUBLICATIONS
Ginebra MP et al . ; Calcium phosphate cements as bone drug delivery systems: A review, J Contr ReI 2006, 113: 102- 110.
Morris C, et al . ; The soybean isoflavone genistein induces differentiation of MG63 human osteosarcoma osteoblasts. J. Nutr. 136, 1-5, 2006
Ishimi et al . : Selective effects of genistein, a soybean isoflavone, on B-lymphopoiesis and bone loss caused by estrogen deficiency, Endocrinology 1999, 140 (4) : 1893- 1900
Dong Z. et al . : Alginate/gelatine blend films and their properties for grug controlled release, J Mem Sci, 2006; 280: 37-44
Teng S. et al . : The effect of alginate addition on the structure and morphology of hydroxyapatite/gelatine nanocomposites, Comp Sci Tech 2006; 66: 1532-1538
Ginebra MP et al . : Mechanical and rheological improvement of a calcium phosphate cement by the addition of a polymeric drug, J Biomed Mater Res 2001; 57(1) : 113-118,
Khairoun et al . : Some factors controlling the injectability of calcium phosphate bone cements, J Mater Sci Mater Med 1998; 9: 425-428
Andrianj otovo H. et al . : Effects of polysaccharadies on the cement properties in the monocalcium phsosphate/β- tricalcium phosphate system, Innov Tech Biol Med 1995; 16S (1) : 140-147,
Leroux L. et al . : Effects of various adjuvants (lactic acid, glycerol and chitosan) on the injectability of a calcium phosphate cement, Bone 1999; 25(2) : S31-S34
Schramm L.: Emulsions, Foams and Suspensions 2005, Wiley- VCH Weinheim
Claims
1.- Process for obtaining a composite calcium phosphate foam comprising the steps of: a) forming a biopolymer foam by agitating or gas blowing a polymer aqueous solution comprising gelatin, sodium alginate, soybean derived polymer or combinations thereof; b) mixing the biopolymer foam obtained in a) with calcium phosphate cement powder
2.- Process according to claim 1, wherein a synthetic surfactant is added in the polymer solution in step a) before agitation or gas blowing.
3.- Process according to claim 2, wherein said synthetic surfactant is a non-ionic surfactant.
4.- Process according to claim 3, wherein said non-ionic surfactant is Pluronic, Span or Tween 80, preferably Tween 80.
5.- Process according to claim 4, wherein said Tween 80 is in a concentration in the solution lower than 10 wt%, preferably lower than 1 wt%, more preferably 0,5 wt%.
6.- Process according to any of preceding claims wherein additives selected from the group consisting of setting accelerants or retardants, nucleating agents, glycerol, lactic acid, citric acid, chitosan, polysaccharides and soluble polymers, are previously added to the calcium phosphate cement powder.
7.- Process according to any of the preceding claims wherein growth factors and/or drugs are added at any of steps a) or b) .
8.- Process according to any of the preceding claims wherein the polymer in the polymer aqueous solution is gelatine alone in a concentration between 1 and 40 weight percent, preferably between 1 and 30 weight percent, more preferably between 1 and 20 weight percent.
9.- Process according to claim 8 wherein gelatine solution is foamed at a temperature between about 450C and about 6O0C, preferably between about 5O0C and about 6O0C, for about 1 minute.
10.- Process according to any of claims 1 to 7, wherein the polymers in said polymer solution are gelatine and soybean derived polymer.
11.- Process according to claim 10, wherein the concentration of soybean derived polymer ranges between 10 and 50 weight percent, preferably lower than 30 weight percent, more preferably about 20 weight percent, and the gelatine concentration ranges between 1 and 30 weight percent .
12.- Process according to claims 10 or 11 wherein the gelatine and soy bean derived polymer containing solution is foamed at a temperature between about 450C and about 6O0C, preferably between about 5O0C and about 6O0C, for about 1 minute.
13.- Process according to any of claims 1 to 7 wherein the polymer in the polymer aqueous solution is sodium alginate alone mixed with a synthetic surfactant according to claim 3 and its concentration ranges between 1 and 10 weight percent, preferably about 2 weight percent.
14.- Process according to claim 13 wherein the sodium alginate is mixed with Tween 80 and foamed at room temperature for about 1 minute.
15.- Composite calcium phosphate foam obtainable by a process according to any of the preceding claims.
16.- Use of the composite calcium phosphate foam according to claim 15 as a biomaterial in bone regeneration and/or scaffold for bone tissue engineering.
17.- Use according to claim 16 wherein said scaffold can be shaped or injected with various geometries and set in physiological conditions, both in vitro and in vivo.
18.- Use of the composite calcium phosphate foam obtainable by a process according to any of the preceding claims 10, 11 or 12 for sustaining a controlled release of soy isoflavones in bony tissues.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP09382046.2 | 2009-04-06 | ||
EP09382046 | 2009-04-06 |
Publications (2)
Publication Number | Publication Date |
---|---|
WO2010116321A2 true WO2010116321A2 (en) | 2010-10-14 |
WO2010116321A3 WO2010116321A3 (en) | 2011-02-24 |
Family
ID=42236926
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/IB2010/051475 WO2010116321A2 (en) | 2009-04-06 | 2010-04-06 | Biopolymer-containing calcium phosphate foam, process for obtaining thereof and use for bone regeneration |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2010116321A2 (en) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012062360A1 (en) * | 2010-11-10 | 2012-05-18 | Stryker Trauma Gmbh | Polymeric bone foam composition and method |
WO2012146865A1 (en) | 2011-04-26 | 2012-11-01 | Universite De Lorraine | Novel multiphasic biomaterials and method for manufacturing same |
EP3160422A4 (en) * | 2014-06-25 | 2018-01-24 | Universitat Politècnica De Catalunya | New single-step manufacturing process for foamed biomaterials |
CN113144276A (en) * | 2021-03-16 | 2021-07-23 | 中山大学 | Composite medicine-carrying bone cement and preparation method and application thereof |
CN114989980A (en) * | 2022-07-08 | 2022-09-02 | 江苏省人民医院(南京医科大学第一附属医院) | Multilayer bone-cartilage composite tissue culture device, preparation method and culture method |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2000007639A1 (en) * | 1998-08-07 | 2000-02-17 | Tissue Engineering, Inc. | Bone precursor compositions |
ES2246726B2 (en) * | 2004-08-12 | 2007-11-01 | Universitat Politecnica De Catalunya | SELF-FRAGABLE AND INJECTABLE CALCIUM PHOSPHATE FOAM |
-
2010
- 2010-04-06 WO PCT/IB2010/051475 patent/WO2010116321A2/en active Application Filing
Non-Patent Citations (9)
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2012062360A1 (en) * | 2010-11-10 | 2012-05-18 | Stryker Trauma Gmbh | Polymeric bone foam composition and method |
US10182973B2 (en) | 2010-11-10 | 2019-01-22 | Stryker European Holdings I, Llc | Polymeric bone foam composition and method |
US11185475B2 (en) | 2010-11-10 | 2021-11-30 | Stryker European Operations Holdings Llc | Polymeric bone foam composition and method |
WO2012146865A1 (en) | 2011-04-26 | 2012-11-01 | Universite De Lorraine | Novel multiphasic biomaterials and method for manufacturing same |
EP3160422A4 (en) * | 2014-06-25 | 2018-01-24 | Universitat Politècnica De Catalunya | New single-step manufacturing process for foamed biomaterials |
CN113144276A (en) * | 2021-03-16 | 2021-07-23 | 中山大学 | Composite medicine-carrying bone cement and preparation method and application thereof |
CN114989980A (en) * | 2022-07-08 | 2022-09-02 | 江苏省人民医院(南京医科大学第一附属医院) | Multilayer bone-cartilage composite tissue culture device, preparation method and culture method |
CN114989980B (en) * | 2022-07-08 | 2023-10-10 | 江苏省人民医院(南京医科大学第一附属医院) | Multi-layer bone-cartilage composite tissue culture device, preparation method and culture method |
Also Published As
Publication number | Publication date |
---|---|
WO2010116321A3 (en) | 2011-02-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1787626B1 (en) | Injectable, self-setting calcium phosphate foam | |
Lodoso-Torrecilla et al. | Calcium phosphate cements: Optimization toward biodegradability | |
Montufar et al. | Foamed surfactant solution as a template for self-setting injectable hydroxyapatite scaffolds for bone regeneration | |
AU2001271209B2 (en) | A composition for an injectable bone mineral substitute material | |
AU2007287334B2 (en) | Macroporous and highly resorbable apatitic calcium-phosphate cement | |
CA2487994C (en) | Hydraulic cement based on calcium phosphate for surgical use | |
Ginebra et al. | Factors affecting the structure and properties of an injectable self‐setting calcium phosphate foam | |
US20100226956A1 (en) | Production of moldable bone substitute | |
WO2010116321A2 (en) | Biopolymer-containing calcium phosphate foam, process for obtaining thereof and use for bone regeneration | |
Ginebra et al. | Injectable biomedical foams for bone regeneration | |
EP3160422B1 (en) | New single-step manufacturing process for foamed biomaterials | |
Hablee et al. | Recent developments on injectable calcium phosphate bone cement | |
Tsuru et al. | Bone cements utilised for the reconstruction of hard tissue: basic understanding and recent topics | |
Dorozhkin | Self-Setting Formulations Calcium Orthophosphate (CaPO4) | |
Lodoso Torrecilla | Fine-Tuning the Degradation and Biological Properties of Calcium Phosphate Cements | |
Khairoun et al. | Incorporation of a controlled-release glass into a calcium phosphate cement | |
JP2007229048A (en) | Tricalcium phosphate-based bone substitute material |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 10718704 Country of ref document: EP Kind code of ref document: A2 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 10718704 Country of ref document: EP Kind code of ref document: A2 |