IPC分类号：
A01N63/00 | A61L27/12 | A61L27/38 | A61L27/58 | C01B25/32 | A61L27/56 | A61F2/28
当前申请(专利权)人：
HISTOCELL, S.L.
原始申请(专利权)人：
HISTOCELL, S.L. (DERIO
当前申请(专利权)人地址：
PARQUE TECNOLOGICO 800, 2, 48160, DERIO (BIZCAIA), SPAIN
发明人：
FONT PEREZ, JULIO | CASTRO FEO, MARIA BEGONA | DEL OLMO BASTERRECHEA, MAITE | GARCIA VAZQUEZ, MARIA DOLORES | RUBIO RETAMA, JORGE | LOPEZ CABARCOS, ENRIQUE | RUEDA RODRIGUEZ, CARMEN | TAMIMI MARINO, FALEH | HAMDAN ALI ALKHRAISAT, MOHAMMAD
摘要：
The present invention is comprised within tissue engineering and, specifically, within bone regeneration. The invention relates to a porous three-dimensional matrix of monetite which is biocompatible, has structured porosity and is predefined and reabsorbable, as well as to the method of synthesis capable of producing said material and the applications thereof. These matrices are a perfect base for cell colonization and proliferation, allowing the application thereof in tissue engineering and bone regeneration as a result of their advantageous properties of biocompatibility, reabsorption, osteoinduction, revascularization, etc.
技术问题语段：
The technical problem addressed in this patent text is the need for a material that can be used as a bone substitute that is reabsorbable, biocompatible, osteoinductive, and can be eliminated without causing harm to the patient. The material should also be able to transfer loads to the new bone, decrease the necessary external support or immobilization time, and be resistant enough to decrease the necessary external support or immobilization time. The material should also be able to induce bone formation and be eliminated without causing harm to the patient.
技术功效语段：
The patent describes the creation of matrices that have tiny pores that increase their surface area and the contact with osteoblasts, which are important for bone growth. These matrices are made from Monetite and have the added benefit of being absorbed by the body over time, preventing them from transforming into a different type of material. They also prevent the growth of certain cells that can damage the bone. This process involves combining the matrices with calcium carbonate to make them more stable and prevent the transformation into a different type of material. Overall, these matrices have the potential to enhance bone growth and help regenerate bone tissues.
权利要求：
1. A composition for bone structure regeneration, wherein said composition comprises a plurality of three-dimensional matrices of monetite with structured porosity, wherein said matrices contain vertical cylindrical channels which longitudinally traverse each matrix from one end to the other, wherein all of the channels that longitudinally traverse each matrix are uniformly-sized cylindrical macropores that are uniformly separated from one another by 0.5 mm ±60μm, said macropores having a diameter of 500μm ±60μm, and wherein in each matrix, none of said cylindrical macropores are connected to each other via another macropore contained within said matrix;
wherein said matrices are cylinders with a base diameter between 2 and 15 mm and a height between 1 and 5 mm, and have a perimetric area from said cylinder edge towards the center thereof of at least 0.5 mm which is free of macropores.
2. The composition of claim 1, wherein the monetite content of the matrices is at least 90%.
3. The composition of claim 2, wherein the monetite content of the matrices is 95%.
4. The composition of claim 2, wherein the monetite content of the matrices is 100%.
5. The composition of claim 1, wherein said matrices are obtained by heat-transforming a precursor material.
6. The composition of claim 5, wherein the precursor material which is heat-transformed into monetite consists of a mixture of a solid phase formed by basic calcium phosphates, acidic calcium phosphates, a pore-inducing agent and a retarder which is set by adding distilled water.
7. The composition of claim 6, wherein the molar ratio of basic phosphate/acidic phosphate is 1.6-1.8, the concentration of pore-inducing agent is 1-20% by weight, that of retarder is between 0.4-0.6% by weight and the (P/L) powder-liquid ratio of the monetite matrix is 3.
8. The composition of claim 7, wherein the molar ratio of basic phosphate/acidic phosphate is 1.785, the concentration of pore-inducing agent is 3-10% by weight and that of retarder is 0.54% by weight.
9. The composition of claim 5, wherein the acidic calcium phosphate is monocalcium phosphate, the basic calcium phosphate is beta-tricalcium phosphate, the pore-inducing agent is calcium carbonate and the retarder is sodium pyrophosphate.
10. The composition of claim 5, wherein the precursor material is Brushite.
11. The composition of claim 1, wherein said three-dimensional matrices are obtained from Brushite.
12. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 10 mm, a height of 5 mm, and 64 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards the center thereof which is free of macropores.
13. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 10 mm, a height of 3 mm, and 64 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards the center thereof which is free of macropores.
14. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 8 mm, a height of 5 mm, and 39 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards the center thereof which is free of macropores.
15. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 8 mm, a height of 3 mm, and 39 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards the center thereof which is free of macropores.
16. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 7 mm, a height of 5 mm, and 28 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards the center thereof which is free of macropores.
17. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 7 mm, a height of 3 mm, and 28 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards the center thereof which is free of macropores.
18. The composition of claim 11, wherein said matrices are cylinders with a base diameter of 5 mm, a height of 3 mm, and 12 cylindrical macropores, respecting a perimetric area of 0.5 mm from said cylinder edge towards center thereof which is free of macropores.
19. The composition of claim 1, wherein said matrices further comprise cells.
20. The composition of claim 19, wherein said cells are mesenchymal cells, osteoblasts, osteoclasts, osteocytes, endothelial cells or combinations thereof.
技术领域：
[0002]The present invention is comprised within tissue engineering and, specifically, within bone regeneration. The invention relates to a porous three-dimensional matrix of monetite which is biocompatible, has structured porosity and is predefined and reabsorbable, as well as to the method of synthesis capable of producing said material and to the applications thereof. These matrices are a perfect base for cell colonization and proliferation, allowing the application thereof in tissue engineering and bone regeneration as a result of their advantageous properties of biocompatibility, reabsorption, osteoinduction, revascularization, etc.
BACKGROUND OF THE INVENTION
[0003]The loss of bone mass and quality is a serious health problem which is even more common in elderly patients.
[0004]The success in the regeneration of a bone defect using three-dimensional materials, which are initially colonized by progenitor cells in vitro, depends to a great extent on the characteristics and structure of the material.
[0005]Biomaterials have been used for almost a century to repair or replace bone segments of the musculoskeletal system.
[0006]The use of autogenous bone grafts, i.e., from the individual himself, is a widely used method for filling bone cavities and for surgical reconstructions. However, there is a limited bone supply and the patient must furthermore be subjected to additional trauma in order to obtain the graft. Another option is donor allografts which also have drawbacks such as a slower neoformation rate, lower osteogenic capacity, reabsorption rate, lower revascularization as well as a higher risk of immunogenic response and transmission of pathogenic agents.
[0007]It is ideal to obtain a material similar to bone, which is biocompatible, does not present adverse biological reactions, is reabsorbable and is gradually degraded as the new tissue is formed, thus progressively transferring the loads to the new bone, preventing a second surgical intervention for removing the implant. A material the degradation products of which are easy to eliminate and non-toxic, which is osteoinductive and induces bone tissue formation is also ideal.
[0008]In the organism, bone degradation and reabsorption are carried out by osteoclasts. They are cells derived from monocytes which are fixed to the surface of the bone. Once fixed, they start releasing protons to the exterior, for the purpose of lowering the pH of the external medium. With this acidic environment, the hydroxyapatite crystals forming part of the mineral component of bone are solubilized. The hydroxyapatite of bone is solubilized in amorphous calcium phosphate particles, which are eliminated by macrophages, or in Ca2+ and PO43− ions which accumulate in the extracellular fluid. These ions diffuse towards the blood capillaries, entering the systemic circulation to be eliminated by urine through the kidney. These released ions can also be reused by osteoblasts to form new bone. Osteoclasts are also in charge of the degradation of the organic phase of bone by means of enzymatic processes.
[0009]The research in new biomaterials for bone repair attempts to reduce the need for bone grafts as much as possible, seeking an artificial substitute which over time is reabsorbed and/or is integrated with the adjacent bone and furthermore serves as fixing in osteoporotic fractures. The mechanical properties of the bone substitute must be as similar as possible to those of spongy bone. The material must furthermore aid in the stability of the fracture and be resistant enough to decrease the necessary external support or immobilization time. Said material must be reabsorbable, biocompatible and osteoinductive, i.e., it must attract mesenchymal cells and other cell types located close to the implant and favor the differentiation thereof into osteoblasts, and also osteoconductive, i.e., it mist act as a mold for the formation of new bone.
[0010]Seeking a similarity with what occurs in the organism, the non-reabsorbable materials used up until now are being substituted in bone implants with reabsorbable materials. These biomaterials do not interfere in the development and growth of the new bone formed, since they are gradually replaced by host tissue. Furthermore, they have a higher biocompatibility, they participate naturally in bone reconstruction and it is not necessary to remove them by means of surgery, after bone regeneration. These materials have to remain for the sufficient time for correct bone regeneration to take place and disintegrate gradually without harming the patient and without intervening in the correct development and growth of the bone.
[0011]The biomaterials which set forming a mineral calcium phosphate are especially interesting in bone regeneration since they resemble the mineral phase of natural bone and are susceptible of bone remodeling and of reabsorption due to their metastable crystal structure.
[0012]The reabsorbable materials which are being used as bone substitutes include calcium phosphates; hydroxyapatite (HAP), tricalcium phosphate (B-TCP) and dicalcium phosphate dihydrate (DCPD) (Stubbs et al., 2004; Schnettler et al., 2004). These materials have an excellent biocompatibility due to their chemical and crystalline similarity to the mineral component of bone, but have difficulties in relation to solubility and reabsorption capacity in vivo.
[0013]Hydroxyapatite (HAP) has been one of those which has aroused the greatest interest. This material is per se the inorganic phase from which bones are formed and it has therefore been widely used in bone regeneration. An example of this are some commercial products such as Interpore 200® Interpore 500®, Cerasorb® and Collagraft®. However, and due to the fact that it has one of the most stable crystal structures, the material has a slow reabsorption.
[0014]HAP is the material having the highest biocompatibility, as it is the most similar one to the crystals formed by bone, but it is not reabsorbable in vivo. The degradation of this material occurs by contact with solutions with a low pH and by phagocytosis. By means of dissolution the amorphous calcium phosphate particles are released, and can be eliminated by macrophages by phagocytosis or be embedded in the new bone formed. Macrophages can dissolve these particles and restore Ca and P to the pool of the organism (Frayssinet et al., 1999; Benahmed et al., 1996). However, it has not been observed that these particles give rise to osteoclast activation (Frayssinet et al., 1999).
[0015]All the studies conducted corroborate the resistance of this material to degradation once it is implanted in the organism, due to its poor solubility at physiological pHs. Implants of this type in animals are reabsorbed by 5.4% in 6 months compared to those based on B-TCP, which are reabsorbed by 85%. (Eggli et al., 1988).
[0016]In humans, the implants made with Bio-Oss (HAP) are considered as non-reabsorbable, since the studies conducted demonstrate that between 3-6 years are needed for them to be reabsorbed due to osteoclast activity (Taylor et al., 2002). The presence of this material in the organism for so much time can interfere in the bone remodeling process, as well as in the osseointegration capacity (Affe et al., 2005; De Boever 2005).
[0017]As a result, this material has traditionally been used in mixtures with organic material as polymers to increase the reabsorption thereof. Examples of these applications are described in U.S. Pat. No. 5,866,155, which describes the incorporation of hydroxyapatite in polylactic matrices, or in U.S. Pat. No. 5,741,329, which is a variation of U.S. Pat. No. 5,866,155 which intends to correct several defects derived from the local acidification of the medium after incorporating cements in the organism.
[0018]To that end, for the purpose of improving the capacity of reabsorption of calcium phosphates and increasing their osteoconductive capacity, crystalline calcium phosphate phases less stable than hydroxyapatite 6, such as B-TCP and DCPD (Brushite), having better solubility and reabsorption in vivo, have been used in recent years.
[0019]B-TCP has more osteoconductivity and a better reabsorption than HAP (Franco et al., 2006). It is considered as a moderately reabsorbable material, in in vivo studies it has been observed that at least one year is needed for its reabsorption in animals and from 6 to 8 months in humans (Wiltfang et al., 2003; Suba et al., 2004). Its degradation increases calcium deposits and this is associated with a higher alkaline phosphatase activity, which enzyme is involved in bone formation (Trisi et al., 2003; Sugawara et al., 2004).
[0020]DCPD is also biocompatible, osteoconductive and the most reabsorbable due to being the most soluble at physiological pHs. This allows new bone to be formed more quickly. It is biodegraded in physiological environments and it is reabsorbed by adjacent cells (Tris et al., 2003). It is proved to be reabsorbed in vivo up to three times quicker than HAP and B-TCP (Herron et al., 2003; Chow et al., 2003; Tas & Bhaduri 2004; Tamini et al., 2006).
[0021]Studies suggest that part of the DPDC material can be converted into HAP after its implantation, which can delay the elimination of the implant by osteoclasts by several weeks (Constanz et al., 1998). This conversion can make the cells acidify the medium and make the biocompatibility of the material decrease together with a reduction in its reabsorption. The addition of Mg and Ca (calcium carbonate) salts or the combination thereof with BTCP can prevent this conversion.
[0022]Using this material it is observed that generation of bone and elimination of the material occur in a balanced manner after the 4th week (Fallet et al., 2006) and the 8th week post-intervention (Constanz et al., 1998). This is important because if the degradation were greater than the synthesis instability and inflammatory reactions would be created.
[0023]Thus, among these calcium phosphates, brushite (DPCD) is one of the materials of greatest interest in bone regeneration. Due to the interesting properties thereof, there are currently brushite cements designed for setting in situ. Thus, for example, U.S. Pat. No. 6,733,582 and US2006213398 claim brushite cements with in situ setting, Chronoss Inject® being an already marketed product of this type. However, this material has a great problem when it is sterilized since it decomposes when it is heated, which makes its appropriate sterilization difficult.
[0024]The state of the art contemplates different publications relating to the sterilization of cements which can be used as bone material substitutes, as well as about the methods used to make said matrices and their sterilization. However, as reflected in patent application JP2004018459, when said cements are sterilized by autoclave, the characteristics of said cements are altered, translating into obtaining bone mineral substitutes which do not meet the characteristics necessary for their use in bone regeneration in terms of reabsorption, stability and colonization and other essential properties.
[0025]As occurs with DPCD, Monetite is reabsorbed in vivo in a similar time and manner. It is gradually dissolved at physiological pHs in the extracellular tissues surrounding the implant and the actual cells colonizing it (endothelial cells, osteoclasts, osteoblasts, macrophages . . . ) would be responsible for the elimination or reuse thereof as occurs in bone.
[0026]Documents such as US20060263443 present Monetite, dicalcium phosphate anhydrous (DCPA), obtained by dehydration of Brushite, in combination with other calcium phosphate biomaterials. Due to the combination, the sterilization results were not acceptable for using these materials in implants and bone regeneration. Additionally, these materials are reaction intermediates and not structures with their own capacity to be used in the technical field of bone regeneration.
[0027]Additionally, for correct bone regeneration, it is necessary for the biomaterial to have a suitable porosity allowing cell colonization and proliferation, vascularization, increase of the surface of contact and therefore increase of the surface of interaction with the host tissue which allows the acceleration of bone regeneration. These characteristics must be accompanied by a correct reabsorption rate providing the cells with the time necessary for regeneration.
[0028]Thus, Gbureck, Uwe et al., 2007, relate to Brushite and Monetite implants prepared by means of the three-dimensional printing technique. To achieve said implants, matrices of brushite which are hydrothermally dehydrated, being transformed into Monetite, are obtained first. However, Table 2 of said article shows that the calcium phosphate material defined as Monetite in said article only has a Monetite content of 63%, not specifying the size or the distribution of its porosity, having a destructured porosity. Thus, said structures are not valid for the purposes of the present invention.
[0029]U.S. Pat No. 6605516 presents bone substitutes with a controlled anatomical shape which adjust exactly to the morphology of the injury. Said substitutes are formed by chemically consolidated calcium phosphate cement materials. The invention also relates to porogenic phases and molds which allow obtaining calcium phosphates with macroporous architectures and external geometries by means of using said molds. However, in its particular embodiments, the invention presents Brushite materials, not presenting Monetite materials and the macroporous structures presented therein also not being valid for the object of the present invention. Thus, the present invention provides matrices of monetite (metastable calcium phosphate phase of monetite), with a high thermal stability which allows sterilizing the material by means of autoclaving, thus simplifying the sterilization processes and which, furthermore, due to its specific structural arrangement of pores, which arrangement is obtained as a result of a specific design of the material, involves an improvement of the osteoinductive capacity of materials proposed by the state of the art since it is synthesized in the form of a porous block with defined structured macroporosity characteristics, increasing the specific surface area, as well as the area of contact with the osteoblasts and facilitating the nutrient transport processes for cells, a crucial factor for bone generation, all of this together with the high capacity of reabsorption thereof in the suitable time period for the adjacent cells to colonize the material and be able to replace the reabsorbed material with physiological bone matrix.
[0030]The in vitro degradation of the matrices of the invention does not affect cell proliferation and they are furthermore bioactive, non-cytotoxic, non-mutagenic and hemocompatible.
发明内容：
[0031]In order for a biomaterial to be able to give rise to a stable bone regeneration, the cells of the implant area, osteoblasts from the adjacent bone, mesenchymal stem cells from bone marrow and endothelial cells from the systemic circulation must be capable of simultaneously and homogeneously colonizing the biomaterial. This will allow the formation of a new physiological bone matrix as the biomaterial is gradually resorbed and the development of a new vascular system, which will provide the blood supply necessary for the survival of the new tissue.
[0032]An important property to be taken into account in relation to this aspect is the porous structure, because it affects both the biodegradability, the higher the degree of porosity the better the reabsorption, and cell colonization. The materials must have pore sizes and interconnections allowing the colonization of both endothelial cells (for the formation of new blood vessels) and of bone cells. Furthermore, the microporous and interconnected nature, which allows the diffusion of nutrients and gases and also of the typical metabolites of cell activity. Bone is not a compact material but rather it has different porosities intercommunicated with one another. Systems of interconnected pores communicate solid (cortical) bone with spongy (trabecular) bone (FIG. 16). These porosities range from 100-150 μm in cortical bone to 500-600 μm in spongy bone.
[0033]The present invention presents a new tissue engineering system, intended to regenerate the bone structure by tackling a curative strategy instead of a merely reparative strategy. Said regeneration is applicable for osteoporosis.
[0034]Tissue engineering is considered as a discipline improving, maintaining and repairing pathologies in organs and in tissues. The creation of a system based on tissue engineering involves the integration of viable cells, a biocompatible material designed especially for a biomedical application and signaling molecules regulating the cell activities required at all times of the treatment.
[0035]Thus, the present invention provides matrices with a geometry of non-random, i.e., ordered or predefined porosity, formed by monetite, in the design of which the porosities of bone have been taken into account, so that neovascularization and cell colonization take place. Said material is presented sterilized, ready for its use and as a result of its specific design, it achieves a specific structural arrangement of pores, i.e., a spatial arrangement and spatial configuration of previously established and induced ordered porosity, which involves an improvement of the osteoinductive capacity compared to other calcium phosphates, including other combinations of calcium phosphate which include monetite.
[0036]Said matrices are obtained in the form of a porous block with defined macro-, meso- and microporosity characteristics which increase the specific surface area, as well as the area of contact with the osteoblasts, facilitating the nutrient transport processes for cells, a crucial factor for bone generation.
[0037]The design of these matrices of monetite of the invention has taken into account the characteristic porosities of natural bone, which porosities allow neovascularization and cell colonization.
[0038]The new matrices of the invention are formed by the biomaterial Monetite, a dehydrated DPCD (DPC), suitable for bone regeneration. Said matrices are formed by at least 95%±5% monetite, preferably by 95% monetite and more preferably by 100% monetite. The traces of material correspond to beta-tetracalcium phosphate. The in vitro degradation of this material does not affect cell proliferation and it is furthermore bioactive, non-cytotoxic, non-mutagenic and hemocompatible as shown in Example 4.
[0039]As a result of their design and composition, the matrices of the invention are reabsorbed in the suitable time period for the adjacent cells to colonize the material and be able to replace the reabsorbed material with physiological bone matrix.
[0040]Matrix relates to any three-dimensional structure useful in bone regeneration which allows the cell growth and proliferation of the cells invading it.
[0041]Cells are understood as:[0042]adult mesenchymal stem cells preferably derived from adipose tissue, but they can also be from bone marrow or any other location which has proved to be a source of these cells. These cells can be used differentiated into the osteoblastic or endothelial strain.[0043]Osteoblasts obtained from bone fragments.[0044]Endothelial cells.[0045]Combinations of adult mesenchymal stem cells that are undifferentiated or differentiated into the osteoblastic or endothelial strain, osteoblasts, osteoclasts, osteocytes from bone and endothelial cells.
[0046]Macropores: when the pores have diameters greater than or equal to 100 microns.
[0047]Mesopores: when the pores have diameters less than 100 microns but greater than or equal to 10 microns.
[0048]Micropores: When the pores have a diameter less than 10 microns.
[0049]Amorphous matrix: A matrix having a geometry of random, non-ordered and non-predefined porosity, which does not follow a spatial distribution and spatial configuration of ordered and previously established porosity, regardless of whether said porosity is natural (intrinsic to the material) or induced.
[0050]Structured matrix or matrix with structured porosity: A matrix having a geometry of non-random, ordered or predefined porosity, having a spatial distribution and spatial configuration of previously established and induced ordered porosity. The matrices of the present invention are matrices with structured porosity with a predefined porosity which confers to them a series of ideal properties for their use in bone regeneration.
[0051]Osteoinduction: bone neoformation by apposition to the material, forming a framework for cell proliferation with osteoblastic activity, forming new bone. It is the act or process of stimulating osteogenesis.
[0052]Osteogenesis: generation or development of bone tissue, through the differentiation of mesenchymal cells into osteoblasts.
[0053]Bone regeneration: formation of new bone which, after a remodeling process, is identical to the pre-existing bone. In bone regeneration, a response is generated in which blood vessels, cells and the extracellular matrix are involved. The biomaterial of the invention is applicable in tissue engineering and bone regeneration and, therefore, can be used in the treatment of the following bone pathologies:[0054]Hypertrophic and non-hypertrophic pseudarthrosis[0055]Osteonecrosis[0056]Osteoporosis[0057]Bone defects caused after the removal of a prosthesis, extirpation of a tumor, by biochemical and metabolic disorders or congenital diseases.[0058]Treatment of injuries and traumas[0059]Treatment of bone fractures[0060]Any pathology in which it is necessary to repair bone tissue.[0061]Treatment of maxillofacial bone defects.[0062]Bone augmentation prior to the application of dental implants
[0063]Cell colonization: capacity of the cells to expand on the biomaterial, being capable of proliferating and increasing the cell population until invading the entire matrix. A measurement of the capacity to colonize a matrix is the analysis of the number of cells on the biomaterial over time (data of the proliferation graph).
[0064]Cell adhesion: capacity of the cells to bind to other cells or to a matrix. Adhesion can occur by specific interactions such as electrostatic forces and is regulated by specific proteins referred to as adhesion molecules. The capacity to adhere to a biomaterial can be analyzed by means of viewing the cells arranged on the biomaterial under a microscope. The surface of contact between the cells and biomaterial will be a representative measurement of the affinity of the cells for that biomaterial.
[0065]In a first aspect, the present invention relates to biocompatible three-dimensional matrices with structured porosity formed by porous monetite, hereinafter matrices of the invention, comprising three-dimensional matrices of monetite with structured porosity, corresponding to cylindrical macropores of between 350-650 μm in diameter, uniformly separated from another by between 0.4-0.6 mm. Said monetite has the intrinsic porosity of the material, on which the indicated structured macroporosity is induced.
[0066]In the matrices of the invention, said structured porosity is distributed in the maximum area of the matrix allowing said matrix to stably maintain its mechanical stability. In a particular embodiment, said maximum area is the area remaining after eliminating the outer perimetric area of the matrix, ranging between 0.1 and 0.9 mm in width, preferably 0.5 mm in width.
[0067]Thus, the materials which are used in osteogenesis must imitate the morphology, structure and function of the bone to achieve a correct integration in the host tissue.
[0068]It has been proved that the structure determined by the porosity and the pore diameter of the materials used in bone regeneration affect bone formation both in vitro and in vivo. The pores are necessary for bone tissue formation to occur, since they allow the migration and the proliferation of osteoblasts and mesenchymal cells and also vascularization. Thus, the material of the invention provides the conditions necessary for achieving correct bone regeneration as a result of its porosity characteristics which allow the colonization and proliferation of the cell types necessary for such effect.
[0069]In vitro results carried out with matrices of other materials show that a low porosity stimulates osteogenesis since cell aggregation occurs, which suppresses proliferation by stimulating osteogenesis. These same experiments show that a high porosity does not affect cell adhesion but does increase proliferation since there is an increase of the surface of contact and the transport of oxygen and nutrients is also facilitated (Takahashi et al., 2004). According to these results, osteogenesis is not affected by the pore size but it does increase with a low number of pores.
[0070]In addition, in vivo, an integration and penetration of the cells in the material as well as the vascularization thereof are required for it to be incorporated to the tissue of the individual. A high porosity and pore size such as those provided by the matrices of the invention facilitate these requirements.
[0071]Initially, according to first studies the minimum diameter required for bone formation was considered to be about 100 μm for the cell migration and transport processes to be carried out. However, diameters greater than 300 μm are currently proposed since the presence of these macropores increases bone formation due to the fact that they allow capillary formation therein. Vascularization affects the development of osteogenesis. Pores with small diameters favor hypoxic conditions and do not induce osteogenesis but rather chondrogenesis.
[0072]Thus, long and large tunnel-shaped pores of the matrix of the invention allow the vascularization thereof and the development of osteogenesis.
[0073]Furthermore, the pores with large diameters increase the surface of contact, which also increases the surface of interaction with the host tissue, which will accelerate the degradation performed by macrophages.
[0074]In the case of amorphous matrices, which have a geometry of random porosity, the vascular network which may be formed is irregular in the structure of the biomaterial and will not be able to connect with the vascular network of the bone, such that the implant will not be able to be effectively integrated with the tissue of the recipient.
[0075]However, the porosity structure adopted by the matrices of the present invention takes into account the incorporation of pores with the suitable size for the co-existence of the required cell species and for the formation of a bone and vascular frame in the entire implant and furthermore for the connection with the recipient area to be allowed, so that tissue integration can take place.
[0076]The new design incorporates cylindrical-shaped (tunnel-shaped) 350 μm-650 μm macropores completely traversing the structure of the material for a suitable cell colonization (in terms of different cell types and a sufficient number of each type) of the cells of the adjacent tissues, as well as an integration with the recipient tissue. Furthermore, in the entire structure it contains a network of micropores for a sufficient diffusion of nutrients, gases and waste products of cell metabolism.
[0077]As can be observed in FIG. 13, the advantage in terms of the cell colonization of the matrices of the invention can be shown in direct studies for cell viewing under a scanning electron microscope. However, as shown in FIG. 14, the amorphous biomaterials, which show a destructured and non-predefined distribution of macropores, produced in the process for obtaining the cement of the present invention, have pores which do not connect the internal structure. In other words, the number of macropores is insufficient and their distribution is unsuitable for a suitable colonization of the cells to take place, such cells being for the most part relegated to the surface of the material.
[0078]The success in the process for forming a new bone is directly related to the amount of bone-forming cells involved in the process, as well as in the formation of a consistent vascular network over the entire biomaterial. Thus, as shown in FIG. 14, the matrices of material with structured porosity of the invention, which have an ordered, induced and previously established spatial distribution and spatial configuration of macropores, allow an extensive cell colonization over the entire biomaterial, a greater diffusion of nutrients and of signaling molecules which will determine cell behavior.
[0079]Therefore, the matrices of the invention, with a high percentage of porosity, especially of macroporosity, in which there are pores with large diameters (>300 μm, specifically between 350 and 650 μm, and preferably 500±60 μm) and in the form of continuous tunnels, will increase the osseointegration of the implant after surgery.
[0080]In a second aspect, the present invention relates to the method of synthesis of the matrices of the invention, which comprises forming a matrix of monetite with structured porosity which comprises:[0081]Forming a solid phase, corresponding to a porous matrix of brushite by means of the combined use of pore-inducing agents, retarder and mechanical methods during the setting reaction between an acidic calcium phosphate and a basic calcium phosphate.[0082]Mixing the solid phase with distilled water to give rise to the liquid phase[0083]Applying in the cement obtained in step 2 one or more molds, one of them with cylindrical punches, having a diameter of between 350 and 650 μm, and more preferably 500 μm±60 μm, during the setting to generate in the matrices vertical cylindrical pores of between 350 and 650 μm, and more preferably 500 μm±60 μm in diameter separated by a distance of between 0.4-0.6 mm and more preferably separated by a distance of 0.5 mm±60 μm.[0084]Sterilizing the porous brushite and heat-transforming it into a porous monetite.
[0085]Specifically, in the method of synthesis used, the product obtained in step 1 gives rise to a solid phase which is mixed with distilled water to give rise to a liquid phase. As a preferred embodiment, the invention proposes using beta-tricalcium phosphate as basic calcium phosphate, and calcium monophosphate as acidic phosphate.
[0086]According to the invention, to carry out the mixing, the molar ratio of basic phosphate/acidic phosphate is 1.6-1.8 for a time of approximately 10 minutes, the concentration of pore-inducing agent is 1-20% by weight and that of retarder is between 0.4-0.6% by weight; preferably a molar ratio of basic phosphate/acidic phosphate of 1.785, a concentration of pore-inducing agents 3-10% by weight and that of retarder is 0.54% by weight.
[0087]The molar ratio of basic phosphate/acidic phosphate to carry out the mixing is 1.6-1.8, preferably 1.785, for a time of approximately 10 minutes. Calcium carbonate is added at concentrations between 1-20% by weight, preferably between 3-100. As a retarder of the setting reaction, the invention proposes using sodium pyrophosphate in a proportion of 0.4-0.6 by weight, 0.54% being the preferential option.
[0088]This solid phase thus obtained is mixed with the liquid phase (distilled water) in a (P/L) ratio of 3.
[0089]With respect to acidic and basic calcium phosphates, pore-inducing agents and retarders to be used in the invention, the person skilled in the art knows the different possible compounds and combinations to be used.
[0090]Molds which allow obtaining the matrices of the invention, which have the structured distribution of pores indicated above, are filled with the paste obtained.
[0091]The mold of the invention used to develop the biomaterial relates to any mold having cylindrical punches, the base of which has a diameter of between 350 and 650 μm, and which are separated from one another by between 0.4 and 0.6 mm. Said mold can be constructed in silicone, metal, resistant plastic material or any type of material allowing it to be applied in its use.
[0092]The mold can have any desired shape, depending on the shape and size required to repair a particular bone defect for each patient, the biomaterial obtained always maintaining the typical porosity characteristics of the biomaterial of the invention, i.e., cylindrical macropores of between 350 and 650 μm in diameter, more preferably 500 μm±60 μm in diameter, uniformly separated from another by between 0.4 and 0.6 mm, more preferably 0.5 mm±60 μm, in addition to the intrinsic porosity of the biomaterial.
[0093]Said molds allow obtaining the matrices of the invention, in which the structured porosity is distributed in the maximum area of the matrix allowing said matrix to stably maintain its mechanical stability.
[0094]In a particular embodiment, said molds allow obtaining matrices in which the maximum area in which the structured porosity is distributed is the area remaining after eliminating the external perimetric area of the matrix, between 0.1 and 0.9 mm in width, preferably 0.5 mm in width.
[0095]The invention also contemplates using more than one mold:[0096]A first mold which allows obtaining the matrices of monetite in the desired shape but without the structured porosity[0097]A second mold which in a planar surface has cylindrical punches, with a diameter of between 350 and 650 μm, preferably 500 μm±60 μm, and which are separated from one another by between 0.4 and 0.6 mm, preferably 500 μm±60 μm. Said second mold must be applied after removing the first mold, introducing therein the parts obtained with the first mold. The second mold is covered with a lid as shown in FIG. 1c.
[0098]Thus, the biomaterial of the invention can be presented in the form of pellets, sheets, cylinders, etc., and any other form which is useful for repairing a particular bone defect of a patient.
[0099]In a preferred aspect of the invention, the mold is in the form of a pellet or cylinder with a diameter between 2 and 50 mm, preferably between 2 and 15 mm, and a height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:[0100]with a diameter of 10 mm and a height of 3 to 5 mm, preferably 3 or 5 mm, having 64 punches, or[0101]with a diameter of 8 mm and a height of 3 to 5 mm, preferably 3 or 5 mm, having 39 punches, or[0102]with a diameter of 7 mm and a height of 3 to 5 mm, preferably 3 or 5 mm, having 28 punches, or[0103]with a diameter of 5 mm and a height of 3 mm, having 12 punches
[0104]In all the cases, the punches are cylindrical with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm, and distributed respecting a perimetric area of 5 mm (taken from the edge of the pellet) free of punches.
[0105]One minute after starting the setting of the cement, the latter is placed for approximately 30 minutes in the mold, before its solidification ends, and it is removed, the pores determined by the mold having been formed. Once it has set completely, the matrix of brushite formed is subjected to autoclaving between 120 and 130° C. for 24-25 minutes, its conversion into Monetite, completely sterilized and suitable for use, occurring.
[0106]In another preferred aspect of the invention, a first mold is made of silicone and has cylindrical cavities for pellets or cylinders of the size of the matrices of the invention which are to be manufactured. In a particular embodiment of the invention, said cavities have a diameter between 2 and 50 mm, preferably between 2 and 15 mm, and a height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:[0107]a diameter of 10 mm and a height of 3 to 5 mm, preferably 3 or 5 mm,[0108]a diameter of 8 mm and a height of 3 to 5 mm, preferably 3 or 5 mm,[0109]a diameter of 7 mm and a height of 3 to 5 mm, preferably 3 or 5 mm,[0110]a diameter of 5 mm and a height of 3 mm,
[0111]Said molds are not involved in the formation of the macropores.
[0112]In this aspect of the invention, the second mold is metallic, it has the dimension of each of the previous parts, and at its base it has, uniformly distributed, 500 microns±60 μm cylindrical punches, separated from one another by 500 microns±60 μm, which give rise to the macroporous component of the matrices of monetite, distributed respecting a minimum perimetric area of 0.5 mm (taken from the edge of the pellet) free of punches. In a particular embodiment, said metallic molds have a diameter between 2 and 50 mm, preferably between 2 and 15 mm, and a height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:[0113]a diameter of 10 mm and a height of 3 to 5 mm, preferably 3 or 5 mm, and 64 punches or[0114]a diameter of 8 mm and a height of 3 to 5 mm, preferably 3 or 5 mm, and 39 punches or[0115]a diameter of 7 mm and a height of 3 to 5 mm, preferably 3 or 5 mm, and 28 punches or[0116]a diameter of 5 mm and a height of 3 mm, and 12 punches
in all the cases respecting a minimum perimetric area of 0.5 mm (taken from the edge of the cylinder) free of punches.
[0117]In this case, the process is identical to the previous one, with the difference that immediately after mixing the solid phase and the liquid phase, the first silicone mold is filled. Before the biomaterial ends its setting, the parts are removed from the silicone mold. The parts are subsequently introduced in the metallic mold with punches (being covered with the metallic lid according to FIG. 1c), until the setting ends in a water bath at 37° C. for 30 minutes. Once solidified, they are removed from the metallic mold obtaining the cylindrical parts with the determined porosity. The matrices formed are subjected to autoclaving between 120 and 130° C. for 24-25 minutes, their conversion into Monetite, completely sterilized and suitable for use, occurring. The use of these molds gives rise to monetite pellets with structured porosity. In a particular embodiment, said pellets have a diameter between 2 and 50 mm, preferably between 2 and 15 mm, and a height between 1 and 50 mm, preferably between 1 and 5 mm, and more preferably:[0118]a diameter of 10 mm and a height of 3 to 5 mm, preferably 3 mm or 5 mm, having a uniform distribution of 64 macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm.[0119]a diameter of 8 mm and a height of 3 to 5 mm, preferably 3 mm or 5 mm, having 39 macropores with a diameter of 500 μm±60 μm, separated from another by 500 μm±60 μm.[0120]a diameter of 7 mm and a height of 3 to 5 mm, preferably 3 mm or 5 mm having 28 macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm.[0121]a diameter of 05 mm and a height of 0.3 mm having 12 macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm.
[0122]In all the cases, the monetite pellets have a minimum perimetric area of 0.5 mm (taken from the edge of the pellet) free of macropores which allows them to maintain the conditions of mechanical stability and strength necessary to be used in their applications.
[0123]Thus, the final distribution of macropores in said pellets respects both the minimum perimetric area of 0.5 mm free of macropores, as well as the size and distance between pores (as described above).
[0124]The products of the present invention are applicable in the field of tissue engineering and bone regeneration. Thus the matrices of monetite of the invention, obtained through the defined molds are applicable for cell support and growth and the applications defined above.
[0125]In a particular embodiment, the pellets of the invention are applied in the form of several units (as an assembly of parts), being arranged such that they adapt completely to the space of the bone defect, facilitating the homogeneous entrance of nutrients, gases and cells in the entire area to be repaired, facilitating recovery thereof as a result of said arrangement and preventing the occurrence of necrotic areas.
[0126]In a preferred aspect, the invention relates to the use of the matrices of the invention as a growth support for mesenchymal cells of different origins, including adipose origin, osteoblasts, endothelial cells and combinations of adult mesenchymal stem cells that are undifferentiated or differentiated into the osteoblastic or endothelial strain, osteoblasts, osteoclasts, osteocytes from bone and endothelial cells, for their use in bone regeneration.
[0127]The matrices of Monetite with structured porosity of the invention are reabsorbed in vivo in a longer time and in a similar manner with respect to DCPD, preventing the drawback of their transformation into HA (as shown by Example 10 which compares the matrices with structured porosity of the invention against matrices of brushite made with the structured porosity of the matrices of the present invention). Thus, said matrices will gradually be dissolved at physiological pHs in the extracellular tissues surrounding the implant and the actual cells colonizing them (endothelial cells, osteoclasts, osteoblasts, macrophages . . . ) will be responsible for the elimination or reuse thereof as occurs in bone. Furthermore, their combination with calcium carbonate in the process for obtaining them prevents their transformation into HAP.
[0128]As occurs with DPCD, the reabsorption thereof starts between the 4th and 8th week, a time period which is suitable for the adjacent cells to colonize the material and be able to replace the reabsorbed material with physiological bone matrix. This biodegradability is adjusted to what occurs in the organism, wherein the bone growth in the defects can take place in a time period comprised between 2 and 6 months, depending on the type of bone and on the size of the defect (Francone V. 2004).
[0129]In addition to the biodegradability, other properties such as the roughness and texture of the material of the invention have been taken into account in the study of the matrices. Thus, according to the biological tests conducted on the matrices of porous monetite with structured macroporosity of the invention, an adhesion to the material greater than 95% is demonstrated, where the cells do not change their morphology in contact with the material and colonize the entire surface, communicating with one another as in any functional tissue.
[0130]It must be taken into account that monetite can show very low resistance and elasticity with respect to that of trabecular bone (elasticity 50-100 MPa and compression 5-10 MPa). However, it would be almost impossible to equal the mechanical properties of bone. And it has been demonstrated that it is enough for the material to reach mechanical properties sufficient to support cell growth, since when the cells invade the material, they will form the organic phase of the implant and the mechanical properties will improve. The matrices of porous monetite of the invention meet with this requirement.
[0131]The monetite material is reabsorbable, bioactive, and has characteristics similar to bone. This material allows cell growth both on its surface and inside it, once in the bone defect it will allow the cells (endothelial cells, osteoblasts, osteoclasts . . . ) to form the necessary scaffold which will be connected to the healthy bone. Subsequently, the monetite will gradually be eliminated little by little, without undergoing transformation into hydroxyapatite, due to the action of osteoclasts, and the osteoblasts will gradually synthesize the new mineral phase which will gradually substitute the monetite, completely eliminating the initial defect.
[0132]Thus, a first object of invention relates to a three-dimensional matrix of monetite with structured porosity characterized by having in its structure vertical cylindrical macropores of between 350 and 650 μm in diameter, which longitudinally traverse the matrix from one end to the other, there being a separation of between 0.4-0.6 mm between each macropore. In a particular embodiment, the diameter of the macropores is preferably 500 μm±60 μm. In another particular embodiment, the separation between macropores is preferably 500 μm±60 μm.
[0133]Another object of the invention relates to the matrix of monetite with structured porosity the monetite content of which is at least 90%, preferably 95% and more preferably 100%.
[0134]A following object of the invention is formed by the matrices of monetite with structure porosity characterized by being obtained by heat-transforming a precursor material. In a particular embodiment, said precursor material which is heat-transformed into monetite consists of a mixture of a solid phase formed by basic calcium phosphates, acidic calcium phosphates, a pore-inducing agent and a retarder which is set by adding distilled water. In another particular embodiment, the molar ratio of basic phosphate/acidic phosphate is 1.6-1.8, the concentration of pore-inducing agent is 1-20% by weight, that of retarder is between 0.4-0.6% by weight and the (P/L) proportion is 3. In another particular embodiment, the molar ratio of basic phosphate/acidic phosphate is 1.785, the concentration of pore-inducing agent is 3-10% by weight and that of retarder is 0.54% by weight. In another particular embodiment, the acidic calcium phosphate is monocalcium phosphate, the basic calcium phosphate is beta-tricalcium phosphate, the pore-inducing agent is calcium carbonate and the retarder is sodium pyrophosphate. In another particular embodiment, the precursor material is Brushite.
[0135]Another object of the invention is formed by the three-dimensional matrices of monetite with structured porosity according to the previous claims characterized in that they can adopt any type of shape required to repair a particular bone or tissue defect. In a particular embodiment, said matrix consists of a cylinder with a base diameter between 2 and 50 mm and with a height between 1 and 50 mm. In another particular embodiment, said cylinder has a base diameter between 2 and 15 mm and a height between 1 and 5 mm. In another particular embodiment, said cylinder has a minimum perimetric area of 0.5 mm free of macropores. In other particular embodiments, the cylinder has:[0136]a diameter of 10 mm, a height of 5 mm, and 64 cylindrical macropores with a diameter of 500 μm±60 μm, uniformly separated from one another by 500 μm±60 μm which longitudinally traverse the matrix.[0137]a diameter of 10 mm, a height of 3 mm, and 64 cylindrical macropores with a diameter of 500 μm±60 μm, uniformly separated from another by 500 μm±60 μm which longitudinally traverse the matrix.[0138]a diameter of 8 mm, a height of 5 mm, and 39 cylindrical macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm which longitudinally traverse the matrix.[0139]a diameter of 8 mm, a height of 3 mm, and 39 macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm which longitudinally traverse the matrix.[0140]a diameter of 7 mm, a height of 5 mm, and 28 macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm which longitudinally traverse the matrix.[0141]a diameter of 7 mm, a height of 3 mm, and 28 macropores with a diameter of 500 μm±60 μm, separated from one another by 500 μm±60 μm which longitudinally traverse
具体实施方式：
[0195]The following examples serve to illustrate but do not limit the present invention.
Example 1
Method of Synthesis of the Matrices of the Invention
[0196]To synthesize the matrices of the invention, a solid phase was mixed with double-distilled water (liquid phase).
[0197]The solid phase comprises but is not limited to an acidic calcium phosphate, a basic calcium phosphate, a pore-inducing agent such as calcium carbonate and a setting retarder such as sodium pyrophosphate.
1.1 Preparation of the Solid Phase
[0198]The solid phase of the calcium cement is made up of a basic calcium phosphate and an acidic calcium phosphate. The basic calcium phosphate is beta-tricalcium phosphate (β-TCP) and the acidic calcium phosphate is monocalcium phosphate. The two components are mixed in a molar ratio of 1.785 in mortar by hand for 10 minutes. Calcium carbonate is added at concentrations between 1-20% (weight/weight) preferably between 3-10%. 0.54% (weight/weight) sodium pyrophosphate is used as a retarder of the setting reaction.
[0199]Specifically, to prepare beta-tricalcium phosphate (β-TCP) 34.42 g of DCPD and 10.01 g CC are mixed (in a molar ratio of 2:1) in a glass mortar and homogenized by hand for 15 minutes. The mixture is heated in an oven (Veckstar) at 900° C. for 14 hours. The synthesis of β-TCP occurs according to the reaction:
2CaHPO4.2H2O+CaCO3→Ca3(PO4)2+5H2O+CO2
[0200]The powder is then sieved and the powder having a particle size less than 322 μm is used.
1.2 Preparation of the Liquid Phase and Synthesis of Monetite Sponges
[0201]The liquid phase is formed by distilled or double-distilled water.
[0202]The solid phase formed by 0.8 g of monocalcium phosphate anhydrous, 1.4 g of beta-tricalcium phosphate, 12 mg of sodium pyrophosphate and 110 mg of carbonate is weighed and 0.77 ml of the liquid phase is mixed in a (P/L) powder-liquid ratio of 3 in a glass plate for 30 s.
1.3 Setting Process
[0203]The cement is set for 30 minutes in a water bath at 37° C. The setting reaction occurs according to the reaction:
Ca3(PO4)2+Ca (H2PO4)2+8H2O→4CaHPO4.2H2O
[0204]During the setting reaction the bicarbonate reacts with the hydrogen ions of the medium, decomposing into carbon dioxide, forming cavities and thus generating a spongy matrix of brushite.
1.4 Washing Process
[0205]The biomaterial is then washed several times in distilled water to eliminate remains of acids in the medium until reaching a pH close to 7, which is optimal for the cell growth which will be carried out in subsequent steps.
1.5 Process for Transforming Brushite into Monetite
[0206]Once the set material is obtained by means of the process described above, it is sterilized. The process for said sterilization comprises autoclaving the set material in a temperature range of 120-130° C. for 24-25 minutes. During this process the brushite is transformed into monetite.
[0207]Process for transforming brushite into monetite:
CaHPO4.2H2O→120° C.→CaHPO4+2H2O (gas)
1.6 Method of Synthesis of the Matrix of Amorphous Porous Monetite.
[0208]Once the compounds have been mixed as descried above (Example 1.1 to 1.2), the resulting cement, brushite, is placed on a surface with the shape of interest for the setting and the subsequent sterilization thereof, thus obtaining an amorphous matrix, with little presence of macropores and irregular distribution thereof, as can be observed in FIGS. 6a and b.
1.7 Method of Synthesis of the Matrix of Monetite with Structured Porosity
[0209]After obtaining the cement by means of the process described in Examples 1.1 to 1.2, one minute after starting the setting, the silicone mold shown in FIG. 2 was applied to the cement for 30 seconds. Once the material has set, it is sterilized as described above (Example 1.5).
[0210]The use of different molds allows obtaining materials having cylindrical pores with a mean size of 500±60 μm and which allow connecting the micro- and macropores generated by the pore-inducing agent.
[0211]FIG. 3 shows an example of matrix of monetite with structured porosity produced by means of the process described in the invention. As a result of the generation of carbon dioxide during the setting reaction as well as the application of the mold described above, the resulting material shows a spongy appearance with a given distribution of pores. A sterile monetite biomaterial with structured porosity, which can be used without further treatments as a matrix for cell growth, is thus obtained.
[0212]FIG. 5 shows the diffraction diagram of the samples before and after the heat treatment in the autoclave. It can be observed in FIG. 4 that, in addition to sterilizing the material, the heat treatment causes the crystalline transformation of the structure from brushite to monetite.
Example 2
Specific Production of Specific Monetite Pellets with Structured Porosity
[0213]By way of example and for the purpose of obtaining cements with optimal characteristics, the powder component formed by 0.8 g of monocalcium phosphate anhydrous, 1.4 g of beta-tricalcium phosphate, 12 mg of sodium pyrophosphate and 110 mg of calcium carbonate was mixed for 30 seconds with 0.77 ml of water. One minute after starting the setting, the molds described below were applied to the cement for 30 seconds.
2.1 Use of a Single Mold in the Process for Obtaining Cylindrical Matrices of Monetite with Structured Porosity
[0214]For the specific performance of this example, silicone molds with the following dimensions and number of punches were used:[0215]a) 1 cm in diameter, 5 mm or 3 mm in height and 64 punches[0216]b) 0.8 cm in diameter, 5 mm or 3 mm in height and 39 punches[0217]c) 0.7 cm in diameter, 5 mm or 3 mm in height and 28 punches[0218]d) 0.5 cm in diameter 3 mm in height and 12 punches
[0219]In all the molds, the punches are cylindrical, with a diameter comprised between 500 μm±60 μm, separated from one another by 500 μm±60 μm, and distributed respecting a perimeter of 0.5 mm (taken from the edge towards the inside of the mode) free of punches. The structure of said punches is that of those depicted in FIG. 2.
[0220]During the setting reaction, as described in Example 1.7, the bicarbonate reacts with the hydrogen ions of the medium, decomposing into carbon dioxide, forming cavities and thus generating a spongy matrix of brushite.
[0221]The biomaterial is then washed several times in distilled water to eliminate remains of acids in the medium until reaching a pH close to 7, which is the optimal one for cell growth.
[0222]The material is subsequently sterilized. In the autoclave sterilization process at 1° C. for 24 minutes, brushite is transformed into monetite, thus obtaining a sterile monetite biomaterial which can be used without further treatments as a matrix for cell growth.
[0223]Thus, the resulting material consists of the specified spongy cylindrical pellets, formed by the biomatrix with structured porosity of the invention, with the dimensions indicated in each case, with macropores distributed homogeneously in said pellets.
[0224]The use of each of the indicated molds allowed obtaining the following matrices with homogeneously distributed cylindrical pores, with a mean pore size of 500 μm±60 μm, separated from one another by 0.5 mm±60 μm, which allow connecting the micro- and macropores generated by the pore-inducing agent:[0225]a) cylindrical pellets of 1 cm in diameter, 0.5 cm or 0.3 cm in height and with 64 macropores (FIG. 4b)[0226]b) cylindrical pellets of 0.8 cm in diameter, 0.5 cm or 0.3 cm in height and with 39 macropores (FIG. 4c)[0227]c) cylindrical pellets of 0.7 cm in diameter, 0.5 cm or 0.3 cm in height and with 28 macropores (FIG. 4d)[0228]d) cylindrical pellets of 0.5 cm in diameter, 0.3 cm in height and with 12 macropores (FIG. 4a)
[0229]As shown in FIG. 4, these monetite pellets of the invention obtained have a perimeter of 0.5 mm (taken from the edge of the pellet towards the inside thereof) free of macropores, allowing them to maintain the conditions of mechanical stability and strength necessary for being used in their applications.
2.2 Use of Two Molds in the Process for Obtaining Cylindrical Matrices of Monetite with Structured Porosity
[0230]For the specific performance of this example, two types of mold, one made of silicone (FIG. 1b) and the other one made of metal (FIG. 1c), were used.
[0231]The silicone mold is used to obtain the Monetite cylinders of suitable size (without intervening in this phase in the formation of the macroporosity).
[0232]To synthesize the silicone mold, cylindrical parts with the same size as the Monetite parts which were to be obtained (FIG. 1a) were first fixed in a glass plate.
[0233]Liquid silicone was then added on the glass plate with the metallic parts, and its polymerization was awaited. Once polymerized, it was removed from the glass plate. The silicone molds obtained have cylindrical cavities of the size of the Monetite units which are to be manufactured (FIG. 1b). Said silicone molds with the cavities of the size of the parts which are to be manufactured do not have punches and, therefore, do not yet contemplate the formation of the macropores.
[0234]7 different silicone molds were obtained, having cylindrical cavities of the following dimensions:[0235]diameter of 10 mm and height of 5 mm or 3 mm,[0236]diameter of 8 mm and height of 5 mm or 3 mm,[0237]diameter of 7 mm and height of 3 or 5 mm,[0238]diameter of 5 mm and height of 3 mm.
[0239]In addition, metallic molds with the dimension of each Monetite part obtained with each of the indicated silicone molds were manufactured. Said metallic molds are made up of two parts, a first part having the punches which give rise to the reproducible macroporous component and a lid (FIG. 1c). Specifically, the dimensions of the manufactured metallic molds were the following:[0240]a) 1 cm in diameter, 0.5 cm or 0.3 cm in height and 64 punches[0241]b) 0.8 cm in diameter, 0.5 cm or 0.3 cm in height and 39 punches[0242]c) 0.7 cm in diameter, 0.5 cm or 0.3 cm in height and 28 punches[0243]d) 0.5 cm in diameter, 0.3 cm in height and 12 punches
[0244]In all the molds, the punches are cylindrical, with a diameter comprised between 500 μm±60 μm, separated from one another by 500 μm±60 μm, and distributed respecting a perimeter of 0.5 mm (taken from the edge towards the inside of the mold) free of punches.
[0245]Once the molds have been manufactured, the monetite parts were created according to the following process:[0246]Firstly, the silicone molds were filled with the product which immediately resulted from mixing the solid phase and the liquid phase.[0247]Secondly, before the biomaterial ended its setting, the parts were removed from the silicone mold. The process is simple since the mold is like a very flexible rubber.[0248]Thirdly, the parts were introduced in the metallic mold with the punches and covered. Said mold is introduced in a water bath at 37° C. for 30 minutes until the end of the setting.[0249]Once completely solidified, they were removed from the metallic mold, obtaining cylindrical parts with the desired porosity.
[0250]The matrices formed were subjected to autoclaving between 120 and 130° C. for 24-25 minutes, their conversion into Monetite, completely sterilized and suitable for its use, occurring.
[0251]The parts obtained have the same porosity and dimensions as the parts obtained in Example 1a (FIG. 4).
Example 3
Comparative Studies Between the Matrices of Monetite with Structured Porosity and Amorphous Monetite
3.1 Microscopic Study
[0252]A comparative assay of microscopic structure of the amorphous matrices and of the matrices with structured porosity of the invention was then carried out. To carry out said assay, scanning electron microscopy techniques by means of known processes for a person skilled in the art were used.
Microscopic Structure of the Matrix of Amorphous Porous Monetite
[0253]The biomaterial arranged in the form of amorphous matrix (FIGS. 6a, b) obtained an uncontrolled porosity. In other words, they show an irregular distribution of macropores, produced during the process for obtaining the cement, described in Examples 1.1 to 1-6. The macropores of the amorphous matrix are cavities in the biomaterial and do not connect the internal structure (FIG. 7).
[0254]In relation to the number and distribution of macropores, the scarcity thereof is observed. The presence of macropores is minimum and they are randomly arranged (FIG. 7).
[0255]Thus, these structures do not favor correct bone regeneration since they do not provide the conditions necessary for correct cell colonization and proliferation.
Microscopic Structure of the Matrix of Structured Porous Monetite
[0256]In contrast, FIGS. 6c and 8 show a matrix of monetite with structured macropores. The scanning microscopy image (FIG. 8) shows the homogeneous distribution of the macropores.
[0257]In contrast to the previous structure, the matrix of monetite with structured porosity will favor correct bone regeneration since it provides the conditions suitable for correct cell colonization and proliferation.
3.2 In Vivo Comparative Study
[0258]One of the most relevant aspects when designing a biomaterial for promoting bone regeneration is developing a structure having a porosity suitable for cell colonization and diffusion of gases and nutrients. Particularly, the macropores (of 100 to 500 μM) allow an optimal medium for the integral colonization of the cells supplied in the matrix, as well as the neovascularization and migration of osteoblasts and osteoclasts of the implant area and the homogeneous formation of new bone in the entire structure provided.
[0259]The biomaterial with structured porosity developed in the present invention has a characteristic macroporous structure which will allow a complete and homogeneous distribution of the osteogenic cells provided in the matrix and furthermore the entrance of cells of the recipient tissue, which will colonize and integrate the new structure, in order to start the resorption process thereof as well as to form new bone matrix which will be gradually deposited on the implant to give rise to new bone, with mechanical and physiological characteristics very similar to the original tissue.
[0260]To determine the advantage formed by the design developed in this work with respect to a non-structured porosity in macropores, a comparative study of the bone regeneration capacity between Monetite biomaterials without macropore structuring and with macroporosity structuring was performed.
[0261]To that end, sheep were used in which a critical defect in the tibia and a stabilization by osteosynthesis techniques were performed. In the defect created, the non-structured Monetite biomaterial was applied in 3 of them and the structured one was applied in the other 3, leaving in all of them the adjacent leg as a control (with formation of the critical defect and stabilization of the fracture but without filling of biomaterial). Before the implantation of the biomaterials, the latter were seeded with an identical number of mesenchymal stem cells from the adipose tissue obtained from the sheep.
[0262]To determine the formation of new bone, a continuous radiographic control and a histological study at 3 and 6 months after the implantation were performed. The results show a clear advantage of the biomaterial with macroporosity with respect to the one which does not have macroporosity. After 3 months from the implantation, a greater colonization of the osteoblasts and osteoclasts of the bone in the entire structure of the macroporous biomaterial, and the homogeneous formation of new bone can be observed. At 6 months, a complete integration of the macroporous material with the design of the invention is observed, with formation of a new vascularization, which will allow the generation of a stable bone, with diffusion of nutrients and oxygen in its entire integrity and without the formation of necrotic areas. However, when the biomaterial does not have a macropore structuring, the formation of new bone tissue restricted to the area peripheral to the implant is observed, leaving the rest of the matrix without cell colonization, either by the previously seeded cells or by those of the recipient tissue, and furthermore the formation of a new vascularization is not induced.
[0263]These results allow concluding that macroporous Monetite has evident advantages with respect to the formation of new bone, due to the colonization of the entire structure of the matrix by the cells of the implantation area, to give rise to a resorption, bone matrix formation and induction of a new vascularization, in a homogeneous manner.
Example 4
In Vitro Biocompatibility Studies
[0264]Before combining the monetite material with structured porosity of the invention with cells, it is necessary to demonstrate that said material is biocompatible.
[0265]The in vitro assays performed were related to cytotoxicity, genotoxicity (mutagenicity) and hemocompatibility, taking into account that the monetite biomaterial with structured porosity of the invention can be considered as an implantable product which will be in permanent contact with bone, the duration of the contact being greater than 30 days.
4.1 Cytotoxicity
[0266]Using cell culture techniques, these assays determine cell lysis (cell death), the inhibition of cell growth and other effects on cells caused by the healthcare products, the materials and/or the extracts thereof.
[0267]By means of this assay it is determined if the material under study, Monetite with structured porosity, is toxic for the cells, affects their proliferation and viability.
[0268]The material analyzed was the matrix of monetite with structured porosity obtained in Example 1, with dimensions of 1 cm in diameter, 5 mm in height and 64 macropores, using PVC as a Positive Control and high-density polyethylene as a Negative Control.
[0269]In relation to the conditions of extraction, since the thickness of the materials is >0.5 mm, 3 cm2 of the material were contacted with 1 ml of the culture medium acting as an extracting agent.
[0270]The cell line used to test the cytotoxicity of the material was the L929 mouse fibroblast line cultured in DMEM culture medium with 10% fetal bovine serum.
[0271]The cytotoxicity and proliferation of monetite with structured porosity were determined by means of the MTT assay. This assay is based on the metabolic reduction of MTT by the mitochondrial enzyme succinate dehydrogenase in a colored compound (formazan) and determines the mitochondrial functional capacity of the cells which have been in contact with the monetite of the invention, according to the positive and negative controls established. The amount of live cells in the culture is thus proportional to the amount of formazan produced and therefore to the amount of absorbance registered by means of a spectrophotometer.
[0272]A commercial cytotoxic standard biomaterial was used as a positive control, and high-density polyethylene and vicryl, also commercial, were used as negative controls. The graphic representation of the proliferation curves obtained for the L929 cells in each of the cases is observed in FIG. 9.
[0273]The results obtained do not show significant differences between the proliferation of the L929 cells in structured Monetite of the invention and in the negative control, demonstrating that the matrix of monetite with structured porosity of the invention is not a cytotoxic biomaterial.
4.2 Mutagenicity
[0274]In the genotoxicity assays, mammalian or non-mammalian cell cultures or other techniques are used to determine gene mutations, changes in the structure or in the number of chromosomes and other DNA or gene alterations caused by the toxicity of the healthcare products, the materials and/or the extracts thereof.
[0275]The in vitro mutagenic potential of the Monetite with structured porosity of the invention was determined by means of the assay referred to as “Mouse Lymphoma Assay”. Said assay is based on quantifying mutations in the thymidine kinase gene in L5178TK+/− mouse lymphoma cells, induced or non-induced after the treatment of these cells with the Monetite biomaterial with structured porosity. The cells deficient in the Thymidine Kinase (TK) gene due to the TK−/− mutation are resistant to the cytotoxic effects of trifluorothymidine (TFT). The cells capable of producing TK are sensitive to TFT, which inhibits the metabolism and stops cell division. Therefore, mutant cells are capable of proliferating in the presence of TFT, whereas normal cells containing at least one allele of the TK gene are not. The assay was performed in 96-well plates and the final result was obtained after visually counting the positive wells (FIGS. 10a and b, in which the growth of a colony of cells is observed) and the negative wells (FIGS. 10c and d, in which no growth is observed). Once the positive and negative wells of each 96-well plate have been counted, a series of formulas established for the assay are applied and the results are expressed in terms of mutation frequencies.
[0276]To carry out the assay, the cells were exposed to the product to be tested in the presence and absence of a suitable metabolic activation system, given that it can occasionally occur that a product to be tested is not mutagenic, but that the metabolites generated in vivo from that product are mutagenic.
[0277]The system most commonly used to simulate the hepatic metabolism in vitro is a post-mitochondrial fraction referred to as S9 to which cofactors are added and which is obtained from rat livers treated with enzyme inducers such as Aroclor 1254. Thus, before the cell treatment, the product to be tested is treated for 2 h with the mixtures referred to as S9, and after that time the cells are treated with the supernatant obtained from this mixture after centrifuging it.
[0278]The following products were used for the treatment of the cells:[0279]As positive controls:[0280]Methyl methanesulfonate (MMS) in the absence of metabolic activation.[0281]3-methylcholanthrene (3-MCA) in the presence of metabolic activation.[0282]As negative controls:[0283]Medium of L5178YTK+/− cells incubated for 24 h.[0284]Medium of L5178YTK+/− cells in the presence of metabolic activation incubated for 24 h.[0285]As product to be tested:[0286]Medium of L5178YTK+/− cells incubated for 24 h with the Monetite biomaterial.[0287]Medium of L5178YTK+/− cells incubated for 24 h with the Monetite biomaterial in the presence of metabolic activation.
[0288]The results obtained (shown in FIG. 11) show that both in the presence and in the absence of metabolic activation it is observed that the negative controls used in the experiment induce a low mutation frequency similar to that of the cells which have been cultivated in the presence of the Monetite with structured porosity. The existence of mutated cells cultured with their culture medium is due to the high spontaneous mutation rate of these cells, thus, this mutation frequency is established as background. In relation to the positive controls, the mutation frequency induced in the L5178YTK+/− cells is clearly higher (about 7 times higher in both cases) than that induced by Monetite or the culture medium. These results demonstrate that Monetite is not a mutagenic biomaterial.
4.3 Hemocompatibility
[0289]These assays evaluate the effects caused on blood or its components by healthcare products or materials which come into contact with blood, using a suitable model or system. The hemolysis assays determine the degree of lysis of the red blood cells and the release of hemoglobin caused by the healthcare products, the materials and/or the extracts thereof in vitro.
[0290]The hemocompatibility of the monetite with structured porosity of the invention was determined by means of a colorimetric assay for determining total blood hemoglobin and hemoglobin released into the plasma when the blood is exposed to monetite. Given that the biomaterial is in solid phase, culture media of cells (osteoblasts and AMSCs) which were in contact for 24 hours with monetite were tested. The results show that the coefficient of variation of the calibration, sample and quality control lines (% CV) is 20% in all the cases (except in the case of calibrator 6) and ⅔ of the values of the quality control line have a percentage of difference with respect to the theoretical one (% PVDF)≦20%, therefore the results of the assay are within the established acceptance criteria.
[0291]The percentages of hemolysis of the compounds used were the following, considering the value of concentration of hemoglobin of 10.19 mg/ml of the blood used as 100% hemolysis:
[0292]Percentage ofCompoundHemolysisPositive control: 1% Triton X-10094Negative control: 40% Polyethylene glycol1.27Medium of bone0Medium AMSCs0Medium of bone + Structured monetite0Medium of AMSCs + Structured monetite0
[0293]These results, shown in FIG. 12, allow concluding that the Monetite with structured porosity of the invention is a hemocompatible biomaterial.
Example 5
Comparative Bioactivity Study Between the Matrix of Amorphous Porous Monetite and the Matrix of Monetite with Structured Porosity
[0294]The bioactivity of a material will depend both on its physicochemical composition and on its structure.
[0295]Thus, in the present example a study is carried out to determine the effect of using the indicated amorphous matrix or matrix with structured porosity on the proliferative capacity of mesenchymal stem cells, one of the cell strains involved in the bone regeneration process together with the osteoblasts of the recipient tissue.
[0296]Once the porous biomatrix has been obtained, as described above, it was washed with culture medium with a pH of 7.4 for one or two hours to hydrate and neutralize the pH (changing the culture medium 2 or 3 times). Adult adipose tissue-derived mesenchymal stem cells (ATMCs) were directly seeded on the material, at a concentration of 0.5.106-6.10° cells per cm2. Two hours after the seeding, culture medium was added until covering the entire material, renewing it every two or three days.
[0297]The cells were cultured in the biomaterial for 7 days, after which the biomatrix to the surface of which the cells had adhered was analyzed by scanning electron microscopy (SEM), in order to observe the adhesion and colonization capacity of said cells on the porous monetite biomaterial.
[0298]The images obtained by SEM (see FIGS. 13a and b), demonstrate that the mesenchymal stem cells are capable of adhering perfectly to the biomaterial, adopting a suitable morphology and that they furthermore establish intercellular contacts, as occurs in a tissue at physiological level (FIGS. 13c and d). As can be observed in FIGS. 13c and d the cells expand perfectly with the biomaterial, interacting maximally therewith and emitting cytoplasmic extensions (filopodia), which increase the surface of contact and increase the level of intercellular contact.
[0299]The biomaterial with structured porosity provides a larger surface to which the cells can adhere, in which they can proliferate and start performing their functions in the bone regeneration process. In other words, they can start creating new bone matrix which will substitute the biomaterial and express signaling molecules which will enhance and direct bone remodeling and neovascularization.
[0300]In contrast, the use of the amorphous matrix as a support for cell growth shows that the random distribution of pores is not suitable for an efficient cell colonization to take place (FIGS. 14a and b), such cells being for the most part relegated to the surface of the matrix since they have a significantly larger size than the microporosity characterizing the biomaterial.
[0301]The results, as shown in FIG. 15, demonstrate that a larger number of cells are quantified in the matrix of monetite with structured porosity. At 24 of culture, the cells in the matrix of monetite with structured porosity proliferate 1.5 times more with respect to those which are in the matrix of amorphous monetite, the proliferation being 1.8 times greater at 48 hours of culture.
[0302]In the matrix of the amorphous monetite, the cells over time give values of proliferation lower than the number of cells arranged at time 0 hours. These cells do not have room for being distributed and are compacted in the macropores without surface continuity, inhibiting the proliferation thereof and being located only in the surface of the material without the possibility of colonizing its interior, they could only be introduced in the small number of macropores which are randomly arranged. These macropores are in the form of hollows which in no case penetrate through the entire structure, which would hinder their interaction with the surrounding tissue in vivo and the arrival of nutrients and oxygen to all the cells. These cells can only be distributed over the surface of the biomaterial. These cells are compacted by lack of space, inhibiting the proliferation thereof and most of them being located only in the surface of the material.
[0303]However, the cells arranged in the matrix of monetite with structured porosity are distributed over all the pores, inside them and over the surface of the material, giving greater values of growth than time 0 hours. These cells are not compacted since they have a larger surface of contact with the material and therefore they do not inhibit the growth thereof.
Example 6
Determination of the Number of Cells to be Implanted Per Surface of Matrix
[0304]There are no studies which allow standardizing or knowing the optimal number of cells in this type of biomaterials, therefore the different investigators carry out their adaptations specifically in order to achieve the maximum clinical result.
[0305]In order for bone regeneration to be successful, the implant has to be integrated in the bone structure of the organism. To that end, the cells of the patient, (endothelial cells, osteoblasts, osteoclasts, macrophages, etc) have to interact with the product and colonize it, together with the supplied cells. In addition, an amount of cells in the product sufficient for the creation of a potent trophic effect, which activates the area and triggers the regenerative process, is necessary.
[0306]In order for the coexistence of cells of the patient and those of the product, a potent trophic effect of the product and a homogeneous cell distribution and diffusion of nutrients, gases and waste products of the metabolism to occur, the biomaterial must supply a large number of cells, but without said cells obturating the porous structure of the biomaterial.
[0307]Furthermore, the cell supply must be considerable since as the biomaterial is gradually degraded, it must be replaced by matrix synthesized by the cells themselves.
[0308]In conclusion, the suitable amount of cells is that which occupies virtually the entire surface of the biomaterial but which does not obturate the porous structure, for the following reasons:[0309]Achieving the sufficient trophic effect to activate the bone regeneration process.[0310]Synthesizing sufficient extracellular matrix to replace the biomaterial.[0311]Allowing the arrival and settlement of cells of the patient involved in bone regeneration, including the endothelial cells in charge of neovascularization.
[0312]To determine the number of cells to be implanted per surface of biomaterial, increasing concentrations of cells were seeded in the biomaterial and the degree of colonization of the structure was observed under SEM. This study also allows determining if the form of seeding used is suitable for the distribution of the cells to be homogeneous.
[0313]The process used consisted of seeding monetite discs of 1 cm in diameter, 0.5 cm in height, and 64 macropores with a diameter of 500 μm, with increasing cell concentrations covering from half a million cells to 6 million per biomaterial (0.5×106-1×106-2×106-3×106-4×106-5×106-6×106). The cells are maintained for 8 days in contact with the biomaterial, to allow the adaptation and settlement thereof. The results are analyzed by SEM.
[0314]The images (FIGS. 17 and 18) indicate that as the cell concentration increases the degree of colonization of the monetite biomaterial with structured porosity of the invention increases, since the capacity of adhesion to the biomaterial is close to 100%. When the lowest dose is applied, the surface of the biomaterial does not show a complete inva