BR102020017697A2 - Process of preparation of bone grafts and bone grafts obtained - Google Patents

Abstract

A process for preparing bone grafts is described from the preparation of a Biosilicate® in (110), in (120) infiltrating Biosilicate® slip into the Biosilicate® of (110), in (130) burning the infiltrated Biosilicate® from ( 120), in (140) infiltrate the Biosilicate® burnt from (130) with Bioglass F18, in (150) burn the product from (140), in (160) repeat the infiltration with Bioglass F18 of (140) and the firing of (150), and in (170) recover the Biosilicate®/Bioglass F18 product with improved compressive strength. The bone grafts obtained showed porosity from 83.0 ± 2%, average cell size of 770 ± 290 μm and compressive strength of 1.0 ± 0.2MPa at porosity of 82.0 ± 1.3%, average cell size of 525 ± 220 μm and compressive strength of 3.3 ± 0.3 MPa as prepared from 35 ppi or 45 ppi PU foams respectively. The bone grafts obtained are used as replacements for trabecular bone and provide consistent mechanical integrity and porosity for regeneration of functional bone tissues.

Description

PROCESS OF PREPARATION OF BONE GRAFTS AND OBTAINED BONE GRAFTS FIELD OF THE INVENTION

[001] The present invention pertains to the field of bioactive synthetic bone grafts, more particularly, to synthetic bone grafts with improved mechanical strength properties over similar bone grafts.

FUNDAMENTALS OF THE INVENTION

[002] Synthetic biomaterials for bone grafting can be classified as first, second and third generation biomaterials and their differences depend on the strength of the response that the implant shows when exposed to body fluids. First-generation biomaterials were created in the 1960s and have since been used in thousands of implants. In general, these are metals and alloys (Ti, stainless steel) and dense or porous ceramics (alumina, zirconia). One of their characteristics is the fact that they are bioinert due to their little or no interaction with the surrounding tissues.

[003] Second generation biomaterials are classified as bioactive or resorbable materials, such as new compositions of ceramics, glasses and glass-ceramics.

[004] A bioactive material is one that provokes a specific biological response at the interface of the material, which results in the formation of a bond between tissues. For example, when bioglasses interact with tissue and body fluids, they rapidly form a layer of hydroxycarbonateapatite (HCA) at the interface between the material and the tissue, increasing the interfacial adhesion of the implant. This mechanism makes it possible to create a stable and lasting interface that increases cellular interaction and controls the growth of mature tissues.

[005] In 1980, bioactive materials such as bioactive glasses, ceramics, glass-ceramics and composites began to have different orthopedic and dental applications. Resorbable materials such as crystalline or amorphous calcium phosphates in contact with body fluids are rapidly dissolved, and the voids formed in the structure are gradually replenished with living tissue. When the material dissolves, the implant begins to lose mechanical strength, generating an unstable interface, causing the patient to be immobilized for a long time. The ideal graft should have a rate of degradation that is synchronized with the formation of new bone tissue.

[006] Although the first and second generation biomaterials constitute important solutions to several clinical problems, for some applications these implants can fail, usually after 10 to 25 years, requiring surgical intervention. Many economic and scientific efforts have been made to improve the performance of these biomaterials without optimal results.

[007] Biological tissues are capable of reacting to dynamic loads or biochemical stimuli; this feature is absent in first and second generation biomaterials, restricting their useful life. As it is necessary to produce a method that generates a more biological response, interest has arisen in the development of third-generation biomaterials, which promote specific cellular responses at the molecular level. These types of biomaterials are characterized by having bioactivity and being absorbable, in addition to presenting better clinical results.

[008] Third-generation Bioglasses allow the activation of genes that stimulate the regeneration of living tissues, conserving the architecture and mechanical properties of the injured bone. Its osteoconduction and osteoinduction are related to the release of ionic dissolution products during the degradation process that generates intracellular and extracellular responses at the interface. This process is linked to the differentiation and proliferation of osteoblasts. When primary human osteoblasts are in contact with ionic dissolution products, seven gene families are activated and regulated, facilitating the formation of new bone tissue. However, despite their high bioactivity, Bioglasses have low mechanical strength, being necessary to find new methods that allow an increase in this.

[009] The main objective of tissue engineering is to repair, replace, preserve or enhance the function of a specific tissue or organ. The ideal bone graft or scaffold for tissue engineering should function as a model for tissue growth in three dimensions, providing an interconnected macroporous network with an appropriate pore size distribution, which promotes vascularization, nutrient delivery and discharge of metabolic waste. Their interaction with cells should encourage adhesion, proliferation, migration and differentiated cell function, but also prevent scar tissue. Ideally, the bone graft or scaffold should be degradable and resorbable at the same rate as tissue regeneration. In addition, it must be strong enough to prevent the collapse of the porous structure and the loss of new tissue structure. Finally, the bone graft or scaffold must be non-toxic to cells and have a high surface area.

[0010] Bioactive glasses can be classified as melt-derived and sol-gel derivatives. Fusion-derived glasses are mainly quaternary, such as Bioglass 45S5 (24.4-Na2O-26.9%CaO46.1SiO2-2.6P2O5, mol%). Bioglass 45S5 can be melted into various powder shapes or sizes and is commercially available as Novabone (USA). These materials have different applications in tissue engineering as they are able to support vascularization in vivo and in vitro; stimulate adhesion, growth and differentiation of osteoblasts; inducing differentiation of mesenchymal cells into osteoblasts; have the ability to unite soft and hard tissues; and in contact with the biological fluid generate a layer of HCA, which is capable of producing a strong bond between Bioglass and human bones. Furthermore, its ionic dissolution products, such as Si4+ , Ca2+ and phosphate, promote the expression of different osteoblast cell genes and other biological responses. The mechanism of osteoinduction is not fully elucidated. However, some studies have shown that during the degradation of Bioglass, silicon, calcium and sodium ions are released together with phosphate groups, which stimulate the formation of bone tissue.

[0011] Despite the high bioactivity of Bioglasses, their poor mechanical properties make their clinical application difficult. A solution to increase its resistance is to induce crystallization in these materials, but it was observed that this process can generate a loss of bioactivity. The best-known bioactive glass is 45S5, which was first synthesized by Larry Hench in the late 1960s and was the first material that showed the ability to form an interfacial bond with host tissue in mice. Its specific composition provides special characteristics, such as the highest index of bioactivity (IB= 12.5), biocompatibility and biodegradability. Being classified as a class A bioactive material for being osteogenic and osteoconductive, when interacting with the body fluid, the surface of Bioglass 45S5 forms a layer of HCA, which increases the activity of osteoblasts. The ionic dissolution products of Bioglass 45S5 can upregulate gene expression, which governs osteogenesis, growth factor production, and angiogenesis. When silicone tubes were coated with Bioglass 45S5, an increase in neovascularization was observed compared to silicone tubes that were not coated with glass.

[0012] The porosity and pore size of bone grafts play a notable role in bone formation in vitro and in vivo. Osteogenesis in vivo was found to be affected by pore size. Pore size affects processes such as vascularization, recruitment and cell penetration. Open and highly interconnected pores are very important for the performance of bone grafts, as they ensure the migration and proliferation of osteoblasts and mesenchymal cells, as well as the growth of blood vessels, which facilitates the supply of nutrients, the supply of oxygen to the cells. and the removal of metabolic wastes. Pores larger than 300 µm promote vascularization and high oxygenation, while pores smaller than 150 µm produce occlusion of cell pores, which hinders cell penetration, production of extracellular matrix and neovascularization of the internal areas of bone grafts. It is known that microporosity, that is, pores smaller than 10 µm, is necessary for capillary growth and interactions between the matrix and cells. Therefore, pore geometry and cell size control are needed to mimic bone characteristics, which can change depending on bone type. In addition, surface roughness improves cell attachment, proliferation and differentiation.

[0013] In bone tissue engineering, suitable pore sizes for bone tissue growth are in the range of 150–900 μm. Pores larger than 900 μm decrease the surface/volume ratio, generating slow tissue neoformation and pores smaller than 150 μm inhibit cell migration and vascularization. Satisfactory porosity and pore sizes are influenced by several factors, such as in vivo bone characteristics, specific biomaterials, cells and cytokines used in vitro and in vivo.

[0014] Although increased porosity and pore size are essential for proper bone growth, these can profoundly affect the mechanical properties of materials due to the structural weakness of bone grafts. Consequently, there is an upper limit to porosity and pore size to prevent the mechanical properties from being severely reduced. For example, Bioglasses are materials that can mimic the extracellular matrix (ECM) and their ion exchange in vivo helps the growth of the HCA layer. For this reason, they are promising materials for bone regeneration, but their highly porous structures can affect their mechanical properties, which can make their wide clinical application difficult. One option to overcome this problem is to coat the surface of bone grafts with a polymer to manufacture strong mechanical structures.

[0015] Bioglasses have been used in different studies related to bone regeneration, using different compositions and porosities. Thus, bone grafts with giant pores, macropores and nanopores were synthesized using bioactive poly(ε-caprolactone) glass that exhibited well-interconnected 3D pore networks. These biomaterials exerted a good influence on bioactivity during bone tissue formation and on cellular activity in in vitro tests. Bioglass 13-93 (6.0Na2O-7.9K2O-7.7MgO-22.1CaO-1.7P2O5- 54.6SiO2, mol%), whose pore diameters were controllable, was also used. The 13-93 glass bone grafts showed excellent microstructure and mechanical response for possible applications in the repair of load-bearing bones.

[0016] Biodegradable bone grafts can be absorbed by the body and, after implantation surgery, their surgical removal is not necessary, for this reason, they have been intensively investigated. This factor must be accompanied by the appropriate degradation rate; thus, while the new bone is formed, the bone graft or temporary scaffold degrades; in other words, these concomitant processes ensure that when the injured tissue is completely regenerated, the bone graft or scaffold is completely degraded. This prevents loss of bone graft or scaffold function due to premature degradation. The rate of degradation is established depending on the application or specific tissue type.

[0017] In general, the consolidation of porous glass-based bone grafts requires heat treatments that induce devitrification. As a consequence, Bioglasses are mainly used in the form of particles to fill bone defects.

[0018] As for the ideal mechanical properties, bone grafts have been used in tissue regeneration to promote the formation and maturation of new tissues or organs, where a balance between temporary mechanical support and mass transport (degradation and cell growth) is essential. ). Therefore, bone grafts must have sufficient mechanical integrity to withstand the implantation procedure and the mechanical forces that are generally experienced during the remodeling process, as well as the patient's normal activities, to prevent the structure from collapsing. Ideally, bone grafts should have adequate initial strength and stiffness, which should be conserved until new tissue is fully formed. For this reason, preferably, the mechanical properties of bone grafts should be similar to those of the surrounding tissue, avoiding load protection (stress shielding) around the bone graft or scaffold and, consequently, bone loss or loosening of the implant. In this sense, this process of mechanical adaptation must be performed especially for bone grafts with an open pore structure, whose bone growth process is carried out within the scaffold. Furthermore, the mechanical properties are related to cell seeding, as certain mesenchymal stem cells (MSCs) differentiate if the stiffness is similar to the respective tissue.

[0019] Metallic biomaterials have been widely used in hard tissue engineering. However, dense metallic materials have a higher Young's modulus value than natural bone, which can cause resorption of surrounding bone tissues, due to the load-protecting effect after implantation. To overcome this disadvantage, it is necessary to develop a porous structure that has a rigidity similar to that of natural bone. Porous metallic bone grafts are often used for application where the tissue supports high loads due to their excellent mechanical properties such as superior fatigue strength, high compressive strength and good machinability. However, these bone grafts have certain deficiencies: as their surfaces are not bioactive, most are not biodegradable and there is the possibility of releasing toxic agents.

[0020] Calcium phosphates, calcium sulfates and bioactive glass have been used as bone regeneration materials. Calcium phosphate and bioactive glasses are capable of inducing the formation, precipitation and deposition of calcium phosphate. On the other hand, the use of bioglass in medical applications represents a great challenge due to its low mechanical properties.

[0021] The compressive strength of natural cancellous bone is in the range of 2 to 12 MPa, and its Young's modulus is around 22 MPa. For this reason, many studies have been developed to manufacture biomaterials that achieve mechanical properties around these values, a requirement for their application in bone tissue engineering.

[0022] For different glass-ceramic bone grafts with porosity lower than 89%, a linear relationship was found between compressive strength and porosity, observing a decrease of 15 to 2 MPa in the value of compressive strength when the porosity of the bone graft or scaffold increases by 10%.

[0023] Some strategies used to increase the low mechanical strength of bone grafts or ceramic scaffolds obtained through the replica technique include coating the surface of bone grafts or scaffolds with different materials, such as natural and synthetic polymers, glasses and composites of these materials. . The mechanical strength of bone grafts or calcium silicate scaffolds increased from 0.32 MPa to 1.45 MPa when their surface was covered with a layer of poly(D, L-lactic acid) (PDLLA). The decrease in porosity after coating was very low, maintaining a porosity above 80% with a significant increase in compressive strength. One of the factors that improved the mechanical properties of bone grafts or scaffolds was that the polymer filled the micropores and microcracks on the surface, preserving the interconnectivity of the bone graft or scaffold.

[0024] The group of researchers from LAMAV (Laboratory of Vitreous Materials) at UFSCar, among which the inventors of the present application, have been developing, since the 2000s, research in order to obtain bioactive materials. These researches resulted in the obtaining of patent protection for some bioactive products that will be listed below.

[0025] Thus, the Brazilian patent PI0300644B1 deals with a process to prepare particulate, bioactive and resorbable biosilicates from glass or fritted plates, the process comprising thermally treating the glassy or fritted plates in one or two heat treatment steps, grinding the crystallized glass obtained and to obtain bioactive crystallized biosilicate powders of desired bioactivity, which in contact with body fluids produces a layer of HCA for restoration of teeth or can be gradually replaced by dental tissue and reabsorbed. For different process conditions, different crystalline phases are obtained in order to prepare a range of bioactive or resorbable silicates that can be used as such or combined in the treatment of different oral problems such as dentin hypersensitivity, dental fissures and xerostomia. The particle size distribution is between 0.1 and 30 microns. The product is marketed under the brand name Biosilicate®.

[0026] Also, the Brazilian patent PI1003676B2, from the same research group that developed the Biosilicate® and from the same authors of the present application, deals with the process of preparing bone grafts or scaffolds through the replica technique. This Brazilian patent deals with suspensions based on Biosilicate® for the preparation of bone grafts or scaffolds. The suspensions comprise, in relation to the total volume of the suspension, from 10-50% of solids consisting of 50 to 98% of porogenic agent, from 2 to 50% of Biosilicate® and from 50 to 90% of liquids consisting of 0.5 -15% of binder and from 85 to 99.5% of liquid medium. In a preparation process, the binder, liquid medium and Biosilicate® are mixed, forming a suspension, the suspension is ground, the porogenic agent is added, the suspension is mixed with the porogenic agent, the mixture is dried into a powder, the powder obtained is sieved, shaped in molds of any geometry, the shaped product is burned and the final bone graft or scaffold product is recovered. The bone grafts obtained by this process present total porosity varying between 65 - 95% and average pore size between 100 and 600 µm.

[0027] Still developed by the same research group at UFSCar, the published Brazilian application BR102013020961 (and the corresponding European patents EP3034480B1 and North American US9731994B1) deals with a vitreous product called F18. This publication deals with a vitreous composition, bioactive vitreous fibers and tissues and articles. A glass composition by amount in % weight SiO2 43-52 is described; Na2O 4-9.5, K20 20.5-32; MgO 0.5-2.5; CaO 15-20; Au0.1-3.5; Ag 0.1-3.5; B203 1.5-4; P205 1-6; ZnO 0.1-3.5; SrO 0.1-3.5. Continuous glass fibers are obtained by downdrawing said molten composition, with lengths from millimeters to kilometers, and diameters between 2 mm and 3 mm. The fibers are covered by collagen and form vitreous tissues. The fabrics form articles for various medical applications. The vitreous tissue obtained is highly bioactive. The composition proposed in this published Brazilian application gives the glass high reactivity and low chemical durability, as is expected for a highly bioactive glass. Its greater vitreous stability allows the glass to be manipulated and undergo heat treatments and/or sintering without uncontrolled crystallization, therefore allowing simple processes to be used, while requiring a low tendency to crystallization.

[0028] The international publication WO2014044666A1 deals with medical implants in the form of bone grafts or titanium dioxide scaffolds. Bone grafts with improved mechanical strength to compression are obtained by annealing the bone graft or scaffold with a low viscosity titanium dioxide suspension in a vacuum infiltration process followed by sintering the bone graft or scaffold. Although this publication deals with increasing the mechanical resistance to compression of the bone graft through vacuum infiltration of a suspension, due to the fact that titanium dioxide (a ceramic formed by covalent bonds between a metal (Ti) and Oxygen) is a powder, the effect of infiltrating a titanium dioxide suspension into the titanium dioxide bone graft is different from that sought in the present invention. Indeed, in the invention, the vacuum infiltration of F18 glass suspension (glass formed by covalent bonds between a semimetal (Si) and Oxygen) is intended to fill defects in the structure of the Biosilicate® bone graft and reduce the porosity of the same flowing between the pores, but without crystallization. Therefore, the mechanical strength improvement mechanism according to the present invention is different from that of the international publication WO2014044666A1.

[0029] In fact, in the international publication WO2014044666A1 a method is described for producing bone grafts or titanium dioxide scaffolds that can be used as bone grafts and whose compressive strength has been improved through various titanium dioxide coatings. Titanium dioxide is a material used in orthopedic implants due to its stability in biological environments and its ability to withstand high physical loads with minimal deformation. In addition, it has high corrosion resistance and good biocompatibility. In addition to a highly interconnected reticular structure, an average cell size greater than 300 µm is required to provide adequate space and permeability for the formation of new bone tissue in a non-resorbable structure such as bone grafts or titanium dioxide scaffolds. Initially, bone grafts or titanium dioxide scaffolds are synthesized using the replica method. As a sacrificial mold, 60 ppi cylindrical polyurethane foams with 10 mm in diameter and 10 mm in height were used. To control the rheological properties of the slips, the pH of the suspensions was kept at 1.5 and under agitation for 2.5 hours at 5000 rpm. First, a slip of titanium dioxide (eg, 65 g of titanium dioxide in 25 mL of water) is applied over the sacrificial mold. After the slip has solidified, the porous mold is fired at a temperature of about 400-550°C at a rate of 0.5°C/min for one hour. Then, the bone graft or scaffold is sintered at a temperature between 1300–1500°C for 2–40 hours at a rate of 3°C/min. This process makes it possible to obtain bone grafts or titanium dioxide scaffolds. Then, a second slip of titanium dioxide (eg, 30–45 g of titanium dioxide in 25 mL of water) is applied over the bone grafts or titanium dioxide scaffolds (sintered for 40 hours) under vacuum. Optionally, the bone graft or scaffold can be subjected to centrifugation to remove excess slip. After the slip solidifies on the bone graft or titanium dioxide scaffold, the bone graft or scaffold is sintered at a temperature of 1500°C for 40 hours at a rate of 3°C/min. This process makes it possible to obtain a titanium dioxide bone graft or scaffold with two titanium dioxide coatings. Optionally, another vacuum infiltration can be performed on bone graft or titanium dioxide scaffold with two titanium dioxide overlays. A third low viscosity titanium dioxide slip (10–20 g titanium dioxide in 25 mL of water) is applied to the bone graft or titanium dioxide scaffold with two titanium dioxide overlays under vacuum conditions (0. 2 mbar for 5 minutes). Optionally, the bone graft or scaffold can be centrifuged to remove excess slip. After the slip solidifies, the bone graft or scaffold is sintered at a temperature of 1500°C for 4 hours at a rate of 3°C/min. Through this procedure, a bone graft or titanium dioxide scaffold with three titanium dioxide coatings is obtained. Alternatively, the order of the second and third titanium dioxide coatings can be reversed. The compressive strength of bone grafts or titanium dioxide scaffolds increased considerably after several titanium dioxide coatings. It was verified that the third coating with titanium dioxide, carried out through the vacuum infiltration process, considerably increased the compressive strength of bone grafts or titanium dioxide scaffolds (3.39 ± 0.77 MPa) in comparison with the bone grafts or scaffolds that were coated only twice with titanium dioxide (1.78 ± 0.52 MPa). However, the interconnectivity of the structures was reduced during the second coating, mainly due to the slip with lower solids content (30 g of titanium dioxide in 25 mL of water). Therefore, this parameter was controlled, as it determines the uniformity and interconnectivity of the pore network when the mechanical integrity of ceramic foams is increased through vacuum infiltration. These bone grafts or scaffolds showed high porosity (88–90%) and adequate cell size (435-450µm). The strategy used in the international patent document WO2014044666A1 to fill the internal porosity of the struts was to increase the consolidation of bone grafts or titanium dioxide scaffolds (bone grafts or scaffolds with only one titanium dioxide coating) by increasing the sintering time. (1500°C). This process allowed the internal porosity of the titanium dioxide struts to be partially filled through better densification of the material. Thus, it was verified that the internal porosity of the struts of bone grafts or titanium dioxide scaffolds was considerably reduced after 20 hours of sintering at 1500°C. However, the increase in compressive strength was very low (< 2 MPa, the minimum value for trabecular bone).

[0030] Therefore, the authors of said international patent document. WO2014044666A1 covered the bone grafts or titanium dioxide scaffolds with various titanium dioxide slips to increase the material's compressive strength. This process was carried out under vacuum conditions to obtain a homogeneous layer on the surface of the bone grafts or scaffolds and to prevent the struts thickness from increasing too much. In addition, the vacuum facilitated the penetration of the slip into the microcracks and microporosities on the surface of the struts, allowing a considerable increase in compressive strength. With this process, the compressive strength of the material was increased without generating a significant decrease in the porosity and interconnectivity of the reticular structure. Another factor that prevented the deposition of a thick layer on the surface of bone grafts or titanium dioxide scaffolds was that, after the second and third infiltration with titanium dioxide, the material was centrifuged to remove excess slip.

[0031] The main objective of the present invention was to increase the compressive strength of bone grafts or Biosilicate scaffolds through several coatings with F18 Bioglass slips under vacuum conditions. This process was carried out to force the glass to penetrate the hollow part of the structure. To facilitate this process, bone grafts or Biosilicate scaffolds were obtained using Biosilicate particles with an average size of 45 µm. Thus, the bone grafts or Biosilicate scaffolds presented a very porous surface that facilitated the penetration of the glass particles. Unlike the invention object of the international publication WO2014044666A1, where the internal porosity of titanium dioxide struts was reduced mainly by increasing the sintering time (20–40 hours), which increased the densification of bone grafts or titanium dioxide scaffolds, in the present invention the internal porosity of the Biosilicate struts was reduced through various vacuum infiltration processes using F18 glass slips, allowing partial filling of the internal porosity (see Figure 2 of the present invention) of the Biosilicate struts.

[0032] The main objective of the present invention was to increase the compressive strength of bone grafts or Biosilicate scaffolds through several coatings with an F18 Bioglass slip.

[0033] In the literature, different types of coatings are reported used to increase the mechanical strength of bone grafts or ceramic scaffolds. In general, synthetic or natural polymers are used to perform this type of coating. In addition, bone grafts or ceramic scaffolds can also be covered using only bioactive glasses. The F18 is ideal for this type of coating, as the Bioglass F18 is sintered at low temperatures and by the viscous flow mechanism. The high stability of this bioglass against crystallization allows it to flow during the process, filling small pores and microcracks. The hollow structure of the bone graft or scaffold is also filled during the infiltration process, becoming solid after sintering. In addition, a smooth, defect-free surface is obtained. All of this contributes to increasing the final mechanical strength of bone grafts or scaffolds. In addition, the F18 glass forms a hydroxycarbonateapatite layer on the surface of the bone graft or Biosilicate scaffold, increasing the bioactivity of the biomaterial, which is very important to enhance the osseointegration and osteogenesis process. This facilitates the processing of the bone graft or scaffold, in contrast to materials such as titanium dioxide which, as they are non-bioactive materials, need surface functionalization to induce the formation of a calcium phosphate layer on the titanium dioxide surface. Another important property of F18 glass and Biosilicate is that they are resorbable materials that can be excreted by the body, avoiding a second surgery to remove them.

[0034] The patent US3090094 describes the basic process of the replica, which consists of impregnating polymeric or natural foams by the ceramic suspension followed by heat treatment, which allows the burning of the organic part and the sintering of the ceramic material, resulting in the replica of the original foam . The optimization of the process steps (choice of polymeric foam, preparation of the ceramic suspension, impregnation, drying and heat treatment) allows the development of materials with desirable characteristics for specific applications. Flexible polyurethane (PU) foams are widely used, with different cell sizes.

[0035] The US published application US20130330537A1 deals with a process for producing bone grafts by the method of coating a sacrificial mold or polyurethane (PU) mold to obtain porous ceramic materials. The foam used as the sacrificial mold is treated and a suspension is coated into the sacrificial mold, which is sintered to form the porous ceramic material. A second suspension is prepared and used to coat the ceramic material, whereupon the blocked pores are released and the material is dried and sintered to form a final sintered ceramic porous material. The final material is considered to be similar to the structure of trabecular bone and provides consistent mechanical integrity and porosity for regeneration of functional bone tissue.

[0036] The microcracks and irregularities on the surface of bone grafts or ceramic scaffolds obtained by the replica technique are generated by a poor coating of slip on the surface of the PU sponge. Furthermore, during heat treatment, the lag between the thermal expansion of the PU foam and the ceramic coating generates these types of surface defects. Another factor that strongly contributes to the reduction of the mechanical properties of ceramic foams or scaffolds synthesized by the replica method is the presence of voids inside the structures that correspond to the space initially occupied by the sacrificial mold or PU foam.

[0037] Given that bone grafts or ceramic scaffolds obtained through the replica technique have surface defects such as microcracks and microporosities, techniques such as surface covering of bone grafts or scaffolds can be used to improve the surface structure of the struts.

[0038] Thus, it would be interesting if the technique had a process for preparing bioactive and resorbable bone grafts based on Biosilicate® and F18 glass, so that the bone graft obtained presents improved mechanical resistance to compression.

SUMMARY OF THE INVENTION

[0039] In general, the process of the invention for preparing bone grafts or scaffolds useful for trabecular bone implants comprises impregnating, by the replica process, sacrificial molds of polyurethane foams (PU) with between 35 and 55 ppi with suspensions or Biosilicate® slips, sinter the impregnated molds under controlled temperatures, obtaining bone grafts or scaffolds and, in a later step, infiltrating under vacuum and repeatedly sintering the product obtained with suspensions of Bioglass F18. The product obtained is a bone graft or scaffold suitable for application in dentistry, in places where the bone tissue does not support high loads.

[0040] The process of the invention for the preparation of bone grafts or scaffolds of Biosilicate/Bioglass F18 therefore comprises several Steps, from I to VII:

[0041] Step I: Preparation of the bone graft or Biosilicate® scaffold. For that, it starts with the bone graft or Biosilicate® scaffold (object of patent PI0300644B1). For the preparation of this bone graft or scaffold, a PU sponge with a medium cell opening in the range 35-55 ppi is used. Then a Biosilicate® slip or suspension is prepared and the PU foam is dipped in the Biosilicate® slip. The infiltrated foam is removed from the slip and excess slip is removed; the foam containing Biosilicate® particles is dried at room temperature for 24 hours. After drying, the foam undergoes the first firing or sintering process, in stages, at increasing temperatures and with maximum temperatures close to 1000°C. The material obtained is a fully crystalline glass-ceramic and not a bioglass. At the end of the process, there may be micropores and residual cracks. The obtained Biosilicate® bone graft or scaffold has a mechanical strength of less than 0.1 MPa.

[0042] Step II: In this Step, the bone graft or Biosilicate® scaffold from Step I is vacuum infiltrated (-400mmHg to -600mmHg) for 5 to 10 minutes with the same Biosilicate® slip as in Step I. The bone graft obtained in this step is dried at room temperature.

[0043] Step III: In this Step, the bone graft or Biosilicate® scaffold infiltrated in step II is submitted to firing, using the same procedure described in Step I.

[0044] Step IV: In Step IV, the bone graft or Biosilicate® scaffold is infiltrated under vacuum by a slip based on Bioglass F18 (object of Brazilian published application BR102013020961). The procedure for infiltrating the Biosilicate® scaffold with the Bioglass F18 slip is similar to that described in Step II.

[0045] Stage V: In Stage V, the bone graft or Biosilicate® scaffold that was infiltrated with the Bioglass F18 slip is submitted to firing under controlled conditions.

[0046] Stage VI: In Stage VI, Stages IV and V are repeated.

[0047] Step VII: In Step VII the Biosilicate®/Bioglass F18 scaffold bone graft product with improved mechanical resistance to compression is recovered.

[0048] Unlike Biosilicate®, Bioglass F18 sinters at low temperatures and by the viscous flow mechanism. The high stability of this bioglass against crystallization allows it to flow during the process, filling small pores and microcracks. The hollow structure of the bone graft or scaffold is also filled during the infiltration process, becoming solid after sintering. In addition, a smooth, defect-free surface is obtained. All of this contributes to increasing the final mechanical strength of the bone graft or scaffold.

[0049] In this way, a bone graft or scaffold of Biosilicate®/Bioglass F18 is obtained, with mechanical strength greater than 3 MPa, that is, compatible with bone grafts or scaffolds in the form of small blocks on the market. The presence of Bioglass F18 increases the bactericidal effect of the bone graft or scaffold.

[0050] And the bone grafts or scaffolds prepared initially from a polymeric sponge of porosity 45 ppi (pores per inch) at the end of the process of the invention have porosity of 82.0 ± 1.3%, average cell size of 525 ± 220 μm and compressive strength of 3.3 ± 0.3 MPa. This highly porous and interconnected three-dimensional structure mimics the morphology of trabecular bone, promoting cell migration, adhesion, proliferation and differentiation.

[0051] On the other hand, bone grafts or scaffolds prepared initially from a polyurethane sponge (PU) of porosity 35 ppi (pores per inch), obtained at the end of the process of the invention, present porosity of 83.0 ± 2%, average size cell size of 770 ± 290 μm and compressive strength of 1.0 ± 0.2 MPa. The 35 ppi polymeric sponge has a larger cell size which reflects in the final result of compressive strength

[0052] The invention therefore provides a process for the preparation of bone grafts or scaffolds from a Biosilicate® bone graft that is subjected to vacuum coating treatments with Bioglass F18 in order to increase its compressive strength so to make the same applicable as engineering material.

[0053] The invention also provides bone grafts or scaffolds resulting from the process, which have porosity of 82.0 ± 1.3%, average cell size of 525 ± 220 μm and compressive strength of 3.3 ± 0.3 MPa

[0054] These properties allow the bone graft or scaffold to mimic the structure of trabecular bone, facilitating the osteogenesis process. In addition, this biomaterial has not only a high bioactivity (osteoconduction and osteoinduction), but also a compressive strength in the range of trabecular bone values (2–12 MPa), being a biomaterial with high potential to be used in bone regeneration of the trabecular bone. mandible, where bone grafts or scaffolds are not subjected to strong tensions, providing adequate tissue resistance during the healing process.

[0055] Thus, an object of the invention is a process of preparing a bone graft or scaffold useful in the regeneration of trabecular bone. The bone graft or scaffold must have a structure with porosity greater than 80%, with open and highly interconnected pores that facilitate migration, adhesion, proliferation and differentiation of cells, stimulating the formation of new bone tissue.

[0056] Another objective of the invention is a process of preparing a bone graft or scaffold of high bioactivity to facilitate the osteogenesis process.

[0057] Another objective of the invention is a process for preparing a bone graft or scaffold with compressive strength between 1.00 ± 0.2 MPa and 3.3 ± 0.3 MPa, from PU-type sacrificial molds with between 35 and 55 ppi (pores per inch).

[0058] Another object of the invention is a bone graft or scaffold comprising a Biosilicate® structure coated with Bioglass F18 so that the final porosity is comprised between 82% and 83%, and the cell size, between 525 µm and 770 µm so as to achieve compressive strength between 1.00 ± 0.2 MPa and 3.3 ± 0.3 MPa.

[0059] Yet another object of the invention is to use Bioglass F18 as a bone graft coating or scaffold. This is because the Bioglass F18 flows without crystallizing at temperatures above 600ºC, joining the microcracks in the Biosilicate® struts, reducing the size of the pores, but without them being obstructed.

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The attached FIGURE 1 is an illustrative flowchart of the various steps of the bone graft or scaffold preparation process according to the invention.

[0061] The attached FIGURE 2 illustrates through SEM micrographs with 500x magnification the effect of filling the pores of a bone graft or Biosilicate® scaffold (Figure 2A) by the infiltration of Bioglass F18 (Figure 2B) under vacuum according to the process of flowchart of Figure 1. Figure 2C clearly shows the filling of a microcrack with Bioglass F18, indicated by the blue arrow.

[0062] The attached FIGURE 3 illustrates the stress-strain curve for bone grafts or Biosilicate® scaffolds using 35 ppi PU foam.

[0063] Attached FIGURE 4 illustrates the stress-strain curve for BioS-2P/Bioglass F18 bone grafts or scaffolds using 35 ppi PU foam.

[0064] Attached FIGURE 5 is a Bar Graph comparing compressive forces between Biosilicate ® bone grafts and BioS-2P/Bioglass F18 bone grafts or scaffolds using 35 ppi PU foam.

[0065] The attached FIGURE 6 illustrates micrographs obtained by SEM showing the porous surface of the bone grafts or Biosilicate ® scaffolds obtained by infiltration in 45 ppi PU foam of slips prepared using the following conditions: FIGURE 6A milling of the Biosilicate ® slip with 30 mm diameter agate spheres at 500 rpm for 10 minutes. Sintering temperature 950°C. 100x amplification. FIGURE 6B grinding with 30 mm diameter agate beads at 500 rpm for 10 minutes. Sintering temperature: 925°C. 100x amplification. To facilitate the penetration of F18 glass particles during vacuum infiltration processes, struts with highly porous surfaces were generated. Different types of grinding to prepare the Biosilicate® slip were tested to coat the PU foams. Furthermore, the sintering was carried out at different temperatures, as can be seen in Figures 6A and 6B, in order to create a highly porous surface.

[0066] Attached FIGURE 7 illustrates SEM micrographs obtained for bone grafts or scaffolds from 45 ppi PU foams under the following conditions: FIGURE 7A shows SEM micrograph (50x magnification) with highly porous bone graft or scaffold structure interconnected after infiltration of the foam with Biosilicate® slip with a particle size of 45 µm; FIGURE 7B shows SEM micrograph (50x magnification) after infiltration of the structure of FIGURE 7A with successive coatings with Bioglass F18 with particle size of 5 µm according to the process of the invention; FIGURE 7C shows SEM micrograph (500x amplification) showing the hollow structure of bone graft or scaffold while FIGURE 7D shows the same structure (500x amplification) after successive coatings with Bioglass F18, with pore size reduction, increasing the mechanical strength of the graft obtained.

[0067] The attached FIGURE 8 is the stress/strain curves for the bone grafts or scaffolds of BioS-P2/F18 prepared according to the invention from PU foams of 35 and 45 ppi.

[0068] Attached FIGURE 9 shows a bar graph comparing compressive strengths between BioS2P/Bioglass F18 bone grafts or scaffolds synthesized using 45ppi and 35ppi foam; and Biosilicate® bone grafts or scaffolds synthesized using 35 ppi and 45 ppi foam. P = porosity and TC = cell size.

[0069] The attached FIGURE 10 is a graph that shows the comparison between the values of compressive strength, obtained by the Ryshkewitch and Gibson & Ashby models and the experimental value obtained for the BioS2P/Bioglass F18 scaffolds (X = 3.3 MPa). For porosity = 82%, n = 5 and σo = 250 MPa.

DETAILED DESCRIPTION OF THE INVENTION

[0070] According to the invention, the following terms have the meaning as follows:

[0071] Foam: it is a cellular structure composed of a network of solid structures called struts. The struts are the edges and faces of the cells, they are the solid part of the foam that is found at the junctions (plateau borders). Open cell foams (eg reticular foams) consist of interconnected cells that allow a fluid to pass through.

[0072] Biosilicate® bone graft or scaffold: It is a bone graft or scaffold prepared in accordance with the teachings of the Brazilian patent BR0306444B1, of the Applicant and fully incorporated herein by reference.

[0073] BioS-2P is a product of the Biosilicate® family and as such will be used in this report as the equivalent of Biosilicate®.

[0074] Bioglass F18: Bone graft or scaffold prepared according to the teachings of Brazilian application BR102013020961, by the Applicant and fully incorporated herein by reference.

[0075] Suspensions for preparing bone grafts or scaffolds used in obtaining the products of the present invention are in accordance with the teachings of the Brazilian patent BR1003676B2, of the Applicant and hereby fully incorporated by reference.

[0076] The present invention comprises preparing bone grafts or scaffolds from coating and sintering of sacrificial molds. These molds are commercial materials, typically polyurethane (PU) foams with pores per inch between 35 and 55 ppi. In the present invention PU foams with 35 ppi and 45 ppi were used.

[0077] According to the replica process used in the invention, depending on the pore size of the sacrificial mold, the bone graft or scaffold produced will have larger or smaller pores, which is important in the sense that the final product has adequate mechanical strength for the desired application.

[0078] From the experiments that led to the present application, the Applicant found that the pores of bone grafts or scaffolds obtained after sintering PU sacrificial molds with 35 ppi impregnated with Biosilicate® suspensions have relatively low compressive strength (1 .0 ± 0.2) and cell size (TC) of 770 µm, even after successive vacuum impregnations with Bioglass F18, see block diagram in Figure 9.

[0079] On the other hand, the process of the invention using a 45 ppi PU product as a sacrificial mold, as it has smaller pores (greater number of pores per inch), these are filled more easily in the vacuum impregnation step of Bioglass F18, producing bone grafts or scaffolds with compressive strength (3.3 ± 0.3 ) and cell size (TC) of 525 µm, these being suitable ranges for application of the product obtained as a trabecular implant.

[0080] The process of the invention for the preparation of bone grafts or scaffolds comprises Steps I to VII, which will be detailed below. The process is also presented in the form of a flowchart in Figure 1.

Step I:

[0081] According to Figure 1, in Step I, in (110), the preparation of the bone graft or scaffold of Biosilicate® is made according to the Brazilian patent PI0300644B1. For the preparation of this bone graft or scaffold, a polyurethane sponge (PU) with an average cell opening in the range 35-55 ppi ("pores per inch" or pores per inch) is used. In this case, the PU sponge can have different geometric shapes, the geometric shape not being a limiting aspect of the invention. A slip (or “suspension”) of Biosilicate® is then prepared. The composition of Biosilicate® slip is shown in Table 1.

[0082] For the preparation of the slip, Biosilicate® is used in the form of a powder with an average particle size of 45 μm (particle size distribution range: 1-100μm). In addition to Biosilicate®, polyvinylbutyral (PVB) is used as a binder and anhydrous ethyl alcohol as a liquid medium. Eventually it is also possible to use other binders such as PVA (polyvinyl alcohol) and CMC (carboxymethyl cellulose) and liquid media such as isopropyl alcohol and even water, such aspects being not critical for the present process. These materials are added to an agate jar containing agate beads and mixed in a planetary mill at 500 rpm for 30-120 minutes.

[0083] It should be noted that the slip mixing may also occur through other mixing methods, such as, for example, in a pitcher spinner or through a mechanical stirrer, this aspect being not critical for the present process.

[0084] Then the PU foam is dipped in Biosilicate® slip, remaining for 5-10 minutes. The infiltrated foam is removed from the slip and the excess slip is removed mechanically by compression (“squeezing”); this procedure is repeated once more.

• a) Aquecimento a uma taxa de 1-5ºC/min até a temperatura de eliminação (“burn-out”) do ligante (PVB) e da espuma (PU), a 400-450ºC;
• b) Patamar de 4 horas na temperatura de 350-450ºC;
• c) Novo aquecimento a 1-5ºC/min até a temperatura de sinterização do Biosilicato® (900-1.100ºC);
• d) Patamar de 3-5horas na temperatura de 900-1.100ºC; e
• e) Resfriamento até a temperatura ambiente, a uma taxa de 1-5ºC/min.

[0085] The foam containing Biosilicate® particles is dried at room temperature for 24 hours. After drying, the foam undergoes the first firing process. The foam burning, in turn, consists of the following steps:

• a) Heating at a rate of 1-5ºC/min until the burn-out temperature of the binder (PVB) and foam (PU), at 400-450ºC;
• b) Level of 4 hours at a temperature of 350-450ºC;
• c) Reheating at 1-5ºC/min until the Biosilicate® sintering temperature (900-1,100ºC);
• d) Level of 3-5 hours at a temperature of 900-1,100ºC; and
• e) Cooling to room temperature at a rate of 1-5°C/min.

[0086] The sintering process of Biosilicate® occurs at high temperatures (~ 1,000ºC) and by the reaction mechanism in the solid state, as this material is a fully crystalline glass-ceramic and not a bioglass. Therefore, the sintering of Biosilicate® is a slower process, and there may be micropores and residual cracks at the end of the process.

[0087] Through this procedure, the bone graft or scaffold of Biosilicate® (with mechanical strength less than 0.1 MPa) is obtained, to be used in later stages. Due to its low mechanical strength, the bone graft or scaffold must be handled with extreme care.

Stage II

[0088] In the following Step II, in (120), the bone graft or Biosilicate® scaffold is infiltrated under vacuum (-400mmHg to -600mmHg) with a Biosilicate® slip whose composition (Table 1) was described in Step I; however, a Biosilicate® powder with an average particle size of 5 μm is used (particle size distribution range: 0.1-20 μm).

• a) O enxerto ósseo ou scaffold é colocado dentro de um recipiente no interior de uma câmara de vácuo;
• b) O vácuo (-400mmHg a -600mmHg) é aplicado;
• c) A barbotina é vertida sobre o(s) enxerto(s) ósseo(s) ou scaffold(s);
• d) Após 5-10 minutos, o vácuo é removido;
• e) O enxerto ósseo ou scaffold é retirado da câmara de vácuo e o excesso de barbotina é removido dos macroporos com o auxílio de uma pistola de ar comprimido; e
• f) O enxerto ósseo ou scaffold é seco à temperatura ambiente por 12- 24 horas e está pronto para ser utilizado na etapa seguinte.

[0089] For the purposes of the invention, a specific apparatus was built for the infiltration of the bone graft or scaffold under vacuum (not shown); however, other devices can be used as long as the operation is carried out under vacuum. In summary, vacuum infiltration comprises the following operations:

• a) The bone graft or scaffold is placed inside a container inside a vacuum chamber;
• b) Vacuum (-400mmHg to -600mmHg) is applied;
• c) The slip is poured over the bone graft(s) or scaffold(s);
• d) After 5-10 minutes, the vacuum is removed;
• e) The bone graft or scaffold is removed from the vacuum chamber and the excess slip is removed from the macropores with the aid of a compressed air gun; and
• f) The bone graft or scaffold is dried at room temperature for 12-24 hours and is ready to be used in the next step.

Stage III

[0090] In Step III, in (130), the bone graft or Biosilicate® scaffold that was infiltrated again with the Biosilicate® slip is submitted to firing, using the same procedure described in Step I above in this report.

Stage IV

[0091] In Step IV in (140), the bone graft or Biosilicate® scaffold is infiltrated under vacuum by a slip based on Bioglass F18 (bioglass object of patent US9731994B2 corresponding to the Brazilian application BR102013020961).

[0092] The composition of Bioglass F18 slip is shown in Table 2:

[0093] To determine the particle size distribution (PSD) the particle size analyzer (Horiba-LA-93) is used in isopropyl alcohol (99.5%-QHEMIS).

[0094] The preparation of Bioglass F18 slip is similar to the preparation of Biosilicate® slip: Bioglass F18 is used in the form of a powder with an average particle size of 5 μm (particle size distribution range: 0.1- 20 μm). In addition to Bioglass F18, powdered polyvinylbutyral (PVB) is used as a binder and anhydrous ethyl alcohol as a liquid medium.

[0095] Eventually it is possible to use other binders such as PVA (polyvinyl alcohol) and CMC (carboxymethyl cellulose) and liquid media such as isopropyl alcohol and even water, this aspect being not critical for the present process.

[0096] These materials are added to an agate jar containing agate beads and mixed in a planetary mill at 500 rpm for 30-120 minutes.

[0097] The slip mixing may also occur through other mixing methods, such as, for example, in a pitcher spinner or through a mechanical stirrer, this aspect being not critical for the present process.

[0098] The procedure for infiltrating the bone graft or Biosilicate® scaffold with the Bioglass F18 slip is analogous to that described in Step II above in this report.

Stage V

• a) Aquecimento a uma taxa de 1-5ºC/min até a temperatura de eliminação (“burn-out”) do ligante (PVB), a 400-450ºC;
• b) Patamar de 4 horas na temperatura de 400-450ºC;
• c) Novo aquecimento a 1-5ºC/min até a temperatura de sinterização do Biovidro F18 (600-900ºC);
• d) Patamar de 3-5horas na temperatura de 600-900ºC; e
• e) Resfriamento até a temperatura ambiente, a uma taxa de 1-5ºC/min.

[0099] In Step V, in (150), the bone graft or Biosilicate® scaffold that was infiltrated with the Bioglass F18 slip is submitted to firing, using the following procedure:

• a) Heating at a rate of 1-5ºC/min until the burn-out temperature of the binder (PVB), at 400-450ºC;
• b) Level of 4 hours at a temperature of 400-450ºC;
• c) Reheating at 1-5ºC/min until the sintering temperature of Bioglass F18 (600-900ºC);
• d) Level of 3-5 hours at a temperature of 600-900ºC; and
• e) Cooling to room temperature at a rate of 1-5°C/min.

[00100] Unlike Biosilicate®, Bioglass F18 is sintered at low temperatures and by the viscous flow mechanism (or “viscous flow”). The high stability of this bioglass against crystallization allows it to flow during the process, filling small pores and microcracks. The hollow structure of the bone graft or scaffold is also filled during the infiltration process, becoming solid after sintering. In addition, a smooth, defect-free surface is obtained. All this contributes to increasing the final mechanical strength of the bone graft or scaffold.

Stage VI

[00101] In Step VI, in (160), Steps IV (140) and V (150) above are repeated in this report. Optionally, Step VI can be repeated more times, taking into account that the porosity of the scaffold must be greater than 80% and the cell size greater than 300 µm.

Stage VII

[00102] In Step VII in (170), the product of the invention is recovered, bone graft or scaffold, Biosilicate®/Biovidro F18, with a highly porous and interconnected structure and with mechanical strength greater than 3 MPa, that is, compatible with bone grafts or scaffolds in the form of small blocks available on the market. The presence of Bioglass F18 increases the bactericidal effect of the bone graft or scaffold.

[00103] The product of the invention, bone graft or Biosilicate®/Bioglass F18 scaffold must be characterized with the aid of several analytical techniques.

[00104] Scanning Electron Microscopy (SEM) analyzes allowed the observation of the microstructures of the sintered samples obtained by the replica technique. Equipment: Philips FEG (XL-30) from the structural characterization laboratory (LCE-DEMa/UFSCar) and the FEI equipment (PHENOMTM) from the vitreous materials laboratory (LaMaV/UFSCar). The samples were covered with gold for 180 seconds.

[00105] The porosity was calculated through the geometric density. First, for bone grafts or prism-shaped scaffolds, the length, height and width of the prism on its four faces were measured, and for cylindrical samples, the diameter and height were measured, and the mean value was calculated. With these data, the average volume of the different specimens was calculated and the density of each bone graft or scaffold was determined using its weight. The mean density was calculated for each type of bone graft or scaffold. Finally, the average porosity of each type of bone graft or scaffold was determined, using Equation 1, where the Biosilicate® density value is 2.79 g.cm-3

[00106] The average pore size was measured using Image-J software.

[00107] Table 3 below lists the total porosity, average cell size and compressive strength values for bone grafts or Biosilicate scaffolds obtained with 35 ppi foam before and after coating with F18 slip.

[00108] The mechanical tests showed that the compressive strengths of bone grafts or Biosilicate scaffolds are very low (Figure 3 attached), with an average compressive strength of 0.020 ± 0.005 MPa. In addition to the high porosity and high average cell size, another factor that significantly contributed to the decrease in the mechanical strength of bone grafts or Biosilicate scaffolds was the internal porosity generated by the burning of the sponge during sintering. For bone grafts or BioS-2P/Bioglass F18 scaffolds, an average compressive strength of 1.0 ± 0.2 MPa was observed (Figure 4 attached), generating an increase of about 50 times the value found for the grafts. bones or Biosilicate® scaffolds (Figure 5 attached). This increase in compressive strength is related to the different coatings with Bioglass F18 slip. This was able to penetrate the surface pores of the structures and partially infiltrate the hollow structure of the bone grafts or Biosilicate® scaffolds. This increase in mechanical strength is also related to the decrease in total porosity and average cell size of bone grafts or Biosilicate® scaffolds (Figure 5 and Table 3 above).

[00109] The microcracks and irregularities on the surface of the ceramic scaffolds obtained by the replica technique are generated by a poor coating of the slip on the surface of the PU sponge. Furthermore, during the heat treatment, the lag between the thermal expansion of the PU foam and the ceramic coating generates these types of surface defects, which greatly diminish the mechanical properties of the ceramic foams or scaffolds. When the Biosilicate® scaffold was infiltrated with the F18 Bioglass slip, the vacuum allowed the slip to penetrate the microcracks and microporosity on the surface of the struts, which allowed a considerable increase in compressive strength and structural uniformity. The layer of Bioglass F18 deposited on the surface of the Biosilicate® scaffolds increased the thickness of the struts, decreasing the total porosity and the average cell size of the bone grafts or Biosilicate® scaffolds (Figure 5 and Table 3 above), which in turn increased its mechanical integrity. Furthermore, the Bioglass F18 particles were able to penetrate the surface pores of the Biosilicate® scaffolds and partially infiltrate the porous struts of the bone grafts or Biosilicate® scaffolds. Despite the considerable increase in compressive strength (50 times), the minimum desired value was not reached, ie, values in the range of compressive strength of trabecular bone (2-12 MPa). A minimum value of 2 MPa allows the use of this type of biomaterials in bone regeneration processes, in places where the bone is not subject to high stresses, such as the mandible. Furthermore, although pore sizes > 300 µm are required for bone tissue formation and vascularization, a very large pore size is associated with a low rate of bone growth, in addition to considerably compromising the mechanical properties of the scaffold.

• a) Usar um pó de Biosilicato® grosso (45 µm) no primeiro procedimento de infiltração para aumentar a porosidade na superfície dos enxertos ósseos ou scaffolds, facilitando a penetração da barbotina nas subsequentes infiltrações; e
• b) Usar pós muito finos (5 µm) para a preparação das barbotinas usadas nas seguintes infiltrações, facilitando a penetração das barbotinas no interior da estrutura oca dos enxertos ósseos ou scaffolds.

[00110] In order to obtain improved results in the infiltration of slip into the hollow structure of bone grafts, the process of the invention contemplates two measures:

• a) Use a coarse Biosilicate® powder (45 µm) in the first infiltration procedure to increase the porosity on the surface of the bone grafts or scaffolds, facilitating the penetration of the slip in the subsequent infiltrations; and
• b) Use very fine powders (5 µm) for the preparation of the slips used in the following infiltrations, facilitating the penetration of the slips inside the hollow structure of the bone grafts or scaffolds.

[00111] The mean cell size for the PU foam (45 ppi) was 590 ± 170 μm and for the bone grafts or BioS-2P/Bioglass F18 scaffolds it was 525 ± 220 μm. Furthermore, particle size distributions were found in the range of 310-1230 μm and 230-1140 μm, respectively.

[00112] The total porosity of the BioS2P/Bioglass F18 bone grafts or scaffolds synthesized using the 45 ppi foam was 82.0 ± 1.3% (Table 4). This value is very close to that presented by BioS-2P/Bioglass F18 scaffolds obtained with 35 ppi foam, 83%. When comparing the average cell size of the 45 ppi foam (590 μm) and the average cell size of the BioS-2P/Bioglass F18 scaffolds (525 μm) also obtained with the 45 ppi foam, it can be observed that these values are similar. This is because the linear shrinkage during the sintering process was small.

[00113] After coatings with Bioglass F18, the bioglass infiltrated the microcracks on the surface of the bone grafts or Biosilicate® scaffolds, helping to consolidate the structure. This homogeneous layer of Bioglass F18 on the surface of the Biosilicate® scaffolds increased the thickness of the struts, strengthening the structure. In addition, the glass penetrated the interior of the struts, partially filling the internal porosity of the structure.

[00114] Other factors such as smaller pore size (525 μm) and lower porosity (82%) of the Biosilicate® scaffold synthesized with a 45 ppi sponge increased the compressive strength of the Biomaterial.

[00115] Considering that 45 ppi PU foam struts are thinner than 35 ppi PU foam struts, the resulting internal porosity in the struts during foam pyrolysis has a smaller volume, increasing the mechanical stability of the grafts bones or scaffolds.

[00116] A highly porous and interconnected structure with a cell size of 525 μm and porosity above 80% are essential for cell penetration and migration, tissue growth, angiogenesis and transport of nutrients, oxygen and waste.

[00117] It is also verified that the bone graft or scaffold obtained by the process of the invention from PU foam of 45 ppi presents compressive strength useful for applications in bone tissue regeneration in places where the bone graft or scaffold is not subject to to high tensions as in breast lift or vertical augmentation surgery.

[00118] The results of total porosity, average cell size and compressive strength for products according to the invention, obtained from PU foam of 35 and 45 ppi respectively are listed in Table 4 below.

[00119] From the results expressed in Table 4 above, the influence of the average cell size on the performance of the product obtained with regard to compressive strength is clear. In case the starting foam has 35 ppi, less pores per inch, the bone graft or scaffold has an average cell size such that the infiltration performed with Bioglass F18 slip does not reach a pore filling and cell size of the bone graft or scaffold such that the recovered product has adequate compressive strength for the desired applications. On the contrary, the bone graft or scaffold obtained with 45 ppi foam has a smaller average cell size, in the appropriate range so that, after infiltration with Bioglass F18 slip, it presents adequate compressive strength for the desired purpose.

[00120] Figure 8 is a graph showing the good stress/strain curve result for the BioS-2P/Bioglass F18 bone graft or scaffold synthesized using a 45 ppi PU foam. This curve is compared to the stress/strain curve for the BioS-2P/Bioglass F18 bone graft or scaffold synthesized using a 35 ppi PU foam.

[00121] In this regard also Figure 9 is interesting, showing not only the results of Table 4, but additionally, the same parameters measured for the bone graft or scaffold of Biosilicate® without infiltration of slip of Bioglass F18. Figure 9 thus illustrates the influence of the number of pores per inch of the foam (35 or 45 ppi), which is reflected in the average cell size of the bone graft or scaffold obtained, whether only of Biosilicate® or the final product of the present process, after infiltration of the bone graft or Biosilicate® scaffold with Bioglass F18 slip. The combination of these factors then determines the compressive strength of the product.

[00122] To demonstrate that the results obtained during the Applicant's research that led to the present application were in accordance with a theoretical model, Figure 10 shows the comparison between the values calculated using the models of Ryshkewitch and Gibson & Ashby and the experimental data obtained for the BioS-2P/Bioglass F18 scaffolds (45 ppi foam), when n = 5, σo = 250 MPa and the porosity is 82%.

[00123] As can be seen in Table 5, the experimental value was close to the theoretical value. However, it was smaller, as the Ryshkewitch model is for dense materials and the modified Gibson & Ashby model assumes that the structure is not hollow in the central region of the struts. For these reasons, the experimental value was expected to be lower. Furthermore, the exact value of n and σo for the BioS-2P/Bioglass F18 scaffolds are unknown. It seems that the experimental data fit both models well when n = 5 and σo = 250 MPa; despite this fact, it would be necessary to determine the exact values of these constants in order to know whether the experimental data really fit the two models well. On the other hand, a possible strategy to increase the mechanical strength of bone grafts or scaffolds would be to use a 60 ppi foam that had a smaller strut size. At the time of burning the foam struts, the hollow part left by the sponge would be smaller due to its smaller size. Although the 60 ppi foam has an average cell size close to (average size = 370µm, size distribution = 140–640µm) to the lower limit (300 µm) required for bone regeneration applications, probably the reduction in cell size during sintering process would be small because of little linear shrinkage, conserving the ideal cell size for bone regeneration. A similar behavior was observed when the foam cell size decreased from 35 ppi to 45 ppi. Therefore, it is expected that a foam with a smaller cell size (370µm) and powders ground with a vibrating mill represent conditions that reduce the internal porosity of the scaffolds after vacuum coatings with Bioglass F18 slip in order to increase their mechanical strength.

[00124] Table 1 lists the compressive strength values determined by the Ryshkewitch and Gibson & Ashby models and the experimental data obtained for the BioS-2P/Bioglass F18 scaffolds synthesized with a 45 ppi sponge. The porosity for the calculated and experimental data was 82%.

[00125] Therefore, the result obtained by the application of the present process is in accordance with the theoretical model of Ryshkewitch and Gibson & Ashby,

[00126] The suitability of the bone graft or scaffold product for its intended use in humans must meet certain criteria, so in vitro tests were performed as described below.

[00127] To perform the in vitro tests, mesenchymal stem cells derived from human adipose tissue (human adipose-derivated stem cells–hASCs) (Sciencell) were used, which were kept in a nitrogen atmosphere in a cryogenic container (Thermo Scientific–model 316306). All cell cultures were performed in a biological safety cabinet (Microzone Corporation-model BK-2-4) under sterile conditions.

[00128] Cells were passaged to 6-well plates for expansion using Mesenchymal Stem Cell Culture Medium (MSCM) (SciencellCanada). The expansion was performed according to the kit manufacturer's recommendations. The plates were incubated for 4 days at 37°C with 5% CO2 in a CO2 incubator (ThermoScientific–Steri-Cycle) until complete confluence. The MSCM was changed every two days. Second, third and fourth passage cells were used for the differentiation process, which were subcultured using MSCM and bone grafts or BioS-2P/Bioglass F18 scaffolds with MSCM, as explained below.

[00129] After complete confluence, the cells were subcultured in a 24-well plate at a density of 1 x 105 cells.cm-2 , using 0.5 mL of MSCM per well and the plates were incubated at 37 °C with 5 % CO2 in incubator for 4 days until 95-100% confluence. These cells were used as controls and others as samples named ion group. In the ion group, the osteogenic medium used to differentiate the cells was placed in contact with bone grafts or BioS-2P/Bioglass F18 scaffolds for 48 hours at 37°C. This process allowed extracting ions from bone grafts or scaffolds.

[00130] In addition, passage of hASCs was also performed on BioS-2P/Bioglass F18 scaffolds (bone graft groups or scaffolds) and expanded using MSCM. First, the bone grafts or BioS-2P/Bioglass F18 scaffolds were previously sterilized in an oven (ThermoScientific model 6520) at 180ºC for three hours. Then, the sterilized glass container with the BioS-2P/Bioglass F18 bone grafts or scaffolds was covered and stored until use. Prior to cell passage, the BioS-2P/Bioglass F18 bone grafts or scaffolds were placed in 24-well plates and left under UV light for 20 minutes. Then, the bone grafts or scaffolds were left for 1 hour with vitronectin solution to facilitate cell adhesion. Finally, the solution was removed and the hASCs were subcultured over the bone grafts or scaffolds at a density of 1 x 105 cells.cm-2 using 1 mL of MSCM to completely cover the bone grafts or scaffolds. The plates were incubated at 37°C with 5% CO2 in an incubator for 4 days until 95-100% confluence. The MSCM was changed every two days. All samples and controls were performed in triplicate.

[00131] Regarding osteogenic differentiation, MSCM was exchanged in all groups for osteogenic differentiation medium for mesenchymal stem cells (MODM) (Sciencell-Canada). This medium was prepared and stored according to the manufacturer's recommendations. 0.5 mL was placed in each well of the controls, 1 mL in each well in the bone grafts or scaffolds group. For the ion group, the osteogenic medium to differentiate the cells was prepared as follows: 10 mL of MODM were left with 1 g of bone grafts or BioS-2P/Bioglass F18 scaffolds at 37°C for 48 hours with 5% of CO2 in an incubator. Then the medium was filtered with a sterile syringe filter (VWR-Ø 0.2 µm) and MODM was added to the filtered medium until reaching a final volume of 50 ml. 0.5 mL of this medium was placed in each well of the ion group. Cells were maintained in culture for 21 days. Several cell viability assays were performed on the cells using the live/dead kit (Invitrogen-Life Technologies), analysis of alkaline phosphatase activity through NBT/BCIP staining (Sigma-Aldrich) and the colorimetric method of paranitrophenol (Abcam-Canada) , mineralization assessment by staining and quantification of alizarin red R (ARS) (Sigma-Aldrich) and gene expression using the RT2 Profiler PCR Arrays Formats C kit (Quiagen).

[00132] The concentration of calcium ions was determined by the photometric method, the concentration of phosphate groups was determined by the UV photometric method and the concentration of sodium ions was determined by an ion-selective electrode at the Maricondi Laboratory (São Carlos/SP-Brazil).

[00133] ALP is a marker in the early stage of osteogenesis, this enzyme catalyzes the hydrolysis of phosphate esters, generating the phosphate groups necessary for the formation and deposition of hydroxyapatite during the osteogenic process. The NBT/BCIP staining assays confirmed the formation of ALP in the controls, in the ion group and in the bone grafts or scaffolds on days 7 and 14. These results were confirmed through the evaluation of the enzymatic activity of ALP, through which it was verified that the amount of ALP was significantly higher in the controls than in the ion group and in the scaffolds on day 7. During this period, the cells of the ion group and the bone grafts or scaffolds were in the process of adaptation, since the osteogenic medium suffered saturation with ionic dissolution products from bone grafts or scaffolds, affecting enzyme production. On day 14, the amount of ALP was significantly higher for bone grafts or scaffolds than for controls and the ion group, which can be attributed to the highly porous and interconnected three-dimensional structure that favors cell migration, proliferation and differentiation. within bone grafts or scaffolds. Furthermore, the hydrophilic surface of bone grafts or scaffolds favors cell adhesion and the ions released by bone grafts or scaffolds activate several genes of the osteogenic process.

[00134] The alizarin red R staining assays allowed observing the formation of calcified nodules in the controls, in the ion group and in the bone grafts or scaffolds on days 7, 14 and 21. The quantification of ARS for the controls and the group of ions showed a more significant deposit of calcium for the ion group than for controls on day 21. This increment can be attributed to ions released into the medium by bone grafts or scaffolds, which can promote the expression of several genes related to osteogenesis.

[00135] Through the analysis of gene expression it was found that bone grafts or scaffolds promote osteogenic differentiation of stem cells derived from human adipose tissue. It is observed that ion groups and bone grafts or scaffolds were able to induce the expression of different members of the BMP superfamily, TGF superfamily receptor, transcription factors, integrin and collagen receptors, all essential for osteogenesis.

Claims (13)

Bone graft preparation process comprising:
a) Prepare in (110) a bone graft or Biosilicate® scaffold from the impregnation of polyurethane foams (PU) with 35-55 ppi (“pores per inch”) with Biosilicate® slip prepared by mixing in a planetary mill at 500 rpm for 30-120 minutes of 20-40% by volume of Biosilicate® powder with an average particle size of 45 μm, 0-5% by volume of a binder selected from PVB, PVA and CMC and 55-80% in volume of a hydroxylated solvent, followed by drying and burning the foam and sintering the Biosilicate® obtained at temperatures ~ 1,000ºC, and separating the Biosilicate® bone graft with mechanical strength lower than 0.1 MPa, said process being characterized by:
b) Infiltrate, at (120), under vacuum (-400 mmHg to -600 mmHg), for 5-10 minutes, the bone graft or Biosilicate® scaffold obtained in a) with the same Biosilicate® slip as in a) except for the average particle size of the Biosilicate® powder is 5 μm, and;
c) In (130), carry out, under the same conditions as in a), the sintering of the infiltrated Biosilicate® obtained in b);
d) Infiltrate, in (140) under vacuum (-400 mmHg to -600mmHg), for 5-10 minutes, the bone graft or Biosilicate® scaffold from step c) with 15-35% by volume of a slip of Bioglass F18 with an average particle size of 5 μm, 0-5% by volume of a binder selected from PVB, PVA and CMC and 60-85% by volume of a hydroxylated solvent, obtaining a bone graft or scaffold infiltrated with Bioglass F18;
e) Burn, in (150), under viscous flow of said Bioglass F18, the infiltrated bone graft from d) under the following firing conditions:

• • Heating at a rate of 1-5ºC/min until the burn-out temperature of the binder at 400-450ºC;
• • Level of 4 hours at a temperature of 400-450ºC;
• • Reheating at 1-5ºC/min to the sintering temperature of Bioglass F18 (600-900ºC);
• • Level of 3-5 hours at a temperature of 600-900ºC; and
• • Cooling to room temperature, at a rate of 1-5ºC/min, recovering a Biosilicate® bone graft infiltrated with Bioglass F18 with a smooth surface;

f) Repeat, in (160) steps d) and e); and
g) Recover, in (170), the bone graft or scaffold of Biosilicate® and Bioglass F18 with compressive strength between 1.0 ± 0.2MPa and 3.3 ±0.3 MPa. Process according to claim 1, characterized by repeating (140) and (150), provided that the porosity of the bone graft obtained is greater than 80% and the cell size is greater than 300 µm. Process according to claim 1, characterized in that the polyurethane foam (PU) has 35 ppi. Process according to claim 3, characterized in that the bone grafts or scaffolds prepared initially from a polyurethane sponge (PU) with a porosity of 35 ppi ("pores per inch") have a porosity of 83.0 ± 2%, an average size of cell of 770 ± 290 μm and compressive strength of 1.0 ± 0.2MPa. Process according to claim 1, characterized in that the polyurethane foam (PU) has 45 ppi. Process according to claim 5, characterized in that the bone grafts or scaffolds prepared initially from a polyurethane sponge (PU) with a porosity of 45 ppi ("pores per inch") have a porosity of 82.0 ± 1.3%, average cell size of 525 ± 220 μm and compressive strength of 3.3 ± 0.3 MPa. Process according to claim 1, characterized in that, in (150), the hollow structure of the bone graft is filled during the infiltration process and becomes solid after sintering. Bone grafts obtained by the process according to claim 1, said grafts being characterized by having porosity of 83.0 ± 2%, average cell size of 770 ± 290 μm and compressive strength of 1.0 ± 0.2MPa. Bone grafts according to claim 8, characterized in that they have a porosity of 82.0 ± 1.3%, an average cell size of 525 ± 220 μm and a compressive strength of 3.3 ± 0.3 MPa. Bone grafts according to claim 8, characterized in that after sintering it presents a solid structure with a smooth and defect-free surface. Bone grafts according to claim 8, characterized in that they are used as bone grafts. Bone grafts according to claim 9, characterized in that they are used for applications in bone tissue regeneration in places where the bone graft or scaffold is not subjected to high stresses. Bone grafts according to claim 12, characterized in that the use is in maxillary sinus lift surgery or vertical bone augmentation of the mandibular region

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