WO2022040771A1 - Procédé de préparation de greffons osseux et greffons osseux obtenus - Google Patents

Procédé de préparation de greffons osseux et greffons osseux obtenus Download PDF

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WO2022040771A1
WO2022040771A1 PCT/BR2021/050372 BR2021050372W WO2022040771A1 WO 2022040771 A1 WO2022040771 A1 WO 2022040771A1 BR 2021050372 W BR2021050372 W BR 2021050372W WO 2022040771 A1 WO2022040771 A1 WO 2022040771A1
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bone
biosilicate
scaffold
bioglass
bone grafts
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PCT/BR2021/050372
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Portuguese (pt)
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WO2022040771A9 (fr
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Claudia PATRICIA MARIN ABADIA
Murilo Camuri Crovace
Edgar Dutra Zanotto
Marina TREVELIN SOUZA
Clever RICARDO CHINAGLIA
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Fundação Universidade Federal De São Carlos
Vetra Pesquisa E Desenvolvimento De Produtos Cerâmicos De Alta Tecnologia Ltda.
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Publication of WO2022040771A9 publication Critical patent/WO2022040771A9/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A61L27/10—Ceramics or glasses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/28—Bones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/02—Inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/02—Inorganic materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/12—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L31/18—Materials at least partially X-ray or laser opaque
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B29—WORKING OF PLASTICS; WORKING OF SUBSTANCES IN A PLASTIC STATE IN GENERAL
    • B29C—SHAPING OR JOINING OF PLASTICS; SHAPING OF MATERIAL IN A PLASTIC STATE, NOT OTHERWISE PROVIDED FOR; AFTER-TREATMENT OF THE SHAPED PRODUCTS, e.g. REPAIRING
    • B29C67/00—Shaping techniques not covered by groups B29C39/00 - B29C65/00, B29C70/00 or B29C73/00
    • B29C67/02—Moulding by agglomerating
    • B29C67/04—Sintering
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C12/00—Powdered glass; Bead compositions
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C3/00—Glass compositions
    • C—CHEMISTRY; METALLURGY
    • C03—GLASS; MINERAL OR SLAG WOOL
    • C03C—CHEMICAL COMPOSITION OF GLASSES, GLAZES OR VITREOUS ENAMELS; SURFACE TREATMENT OF GLASS; SURFACE TREATMENT OF FIBRES OR FILAMENTS MADE FROM GLASS, MINERALS OR SLAGS; JOINING GLASS TO GLASS OR OTHER MATERIALS
    • C03C4/00—Compositions for glass with special properties
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/01—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products based on oxide ceramics
    • C04B35/14—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products based on oxide ceramics based on silica
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/01—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products based on oxide ceramics
    • C04B35/16—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products based on oxide ceramics based on silicates other than clay
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B35/00—Shaped ceramic products characterised by their composition; Ceramics compositions; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/622—Forming processes; Processing powders of inorganic compounds preparatory to the manufacturing of ceramic products
    • C04B35/626—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B
    • C04B35/63—Preparing or treating the powders individually or as batches ; preparing or treating macroscopic reinforcing agents for ceramic products, e.g. fibres; mechanical aspects section B using additives specially adapted for forming the products, e.g.. binder binders
    • C04B35/632—Organic additives
    • C04B35/634—Polymers
    • C04B35/63404—Polymers obtained by reactions only involving carbon-to-carbon unsaturated bonds
    • C04B35/63416—Polyvinylalcohols [PVA]; Polyvinylacetates
    • C—CHEMISTRY; METALLURGY
    • C04—CEMENTS; CONCRETE; ARTIFICIAL STONE; CERAMICS; REFRACTORIES
    • C04B—LIME, MAGNESIA; SLAG; CEMENTS; COMPOSITIONS THEREOF, e.g. MORTARS, CONCRETE OR LIKE BUILDING MATERIALS; ARTIFICIAL STONE; CERAMICS; REFRACTORIES; TREATMENT OF NATURAL STONE
    • C04B38/00—Porous mortars, concrete, artificial stone or ceramic ware; Preparation thereof
    • C04B38/06—Porous mortars, concrete, artificial stone or ceramic ware; Preparation thereof by burning-out added substances by burning natural expanding materials or by sublimating or melting out added substances
    • C04B38/0615—Porous mortars, concrete, artificial stone or ceramic ware; Preparation thereof by burning-out added substances by burning natural expanding materials or by sublimating or melting out added substances the burned-out substance being a monolitic element having approximately the same dimensions as the final article, e.g. a porous polyurethane sheet or a prepreg obtained by bonding together resin particles
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G18/00—Polymeric products of isocyanates or isothiocyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

Definitions

  • 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.
  • Synthetic biomaterials for bone grafting can be classified as first, second and third generation biomaterials and their differences depend on the type of response that the implant presents when exposed to body fluids.
  • Bioglasses are known third-generation materials, and, depending on their composition, activate genes that stimulate the regeneration of living tissues. Its osteoconduction and osteoinduction are related to the release of ionic dissolution products during its degradation process, which generates intra and extracellular responses at its interface. However, despite their high bioactivity, Bioglasses have low mechanical strength, and it is necessary to find new methods to increase it.
  • 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. Its interaction with cells should encourage adhesion, proliferation, and cell migration, and also prevent scar tissue.
  • 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. It should also be non-toxic to cells and have a high surface area.
  • Bioglasses in powder form, have several 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 bond to soft and hard tissues; and in contact with the biological fluid generate a layer of hydroxycarbonateapatite (HCA), which produces a strong bond with human bones. Furthermore, its ionic dissolution products, such as Si 4+ , Ca 2+ and phosphate, promote the expression of different osteoblast cell genes and other biological responses. [006] Despite the high bioactivity of bioglasses, their poor mechanical properties make their clinical application difficult.
  • 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 removal of metabolic wastes.
  • Pores larger than 300 pm promote vascularization and high oxygenation, while pores smaller than 150 pm can be occluded by cells, which hinders their 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 pm, is necessary for capillary growth and interactions between matrix and cells. Therefore, control of pore size and geometry is necessary to mimic bone characteristics. In addition, surface roughness improves cell attachment, proliferation and differentiation. [008] In tissue engineering, suitable pore sizes for bone tissue growth are in the range of 150-900 pm. Pores larger than 900 pm decrease the surface/volume ratio, generating slow tissue neoformation and pores smaller than 150 pm inhibit cell migration and vascularization. Satisfactory porosity and pore sizes are influenced by several factors such as the biomaterials used, bone characteristics in vivo, cells and cytokines used in vitro and in vivo.
  • Bioglass 13-93 (6.0Na2O-7.9K2O-7.7MgO-22,1CaO-1.7P2Os-54.6SiO2, mol%) has also been used for bone grafts whose pore diameters were controllable. These showed an excellent microstructure and mechanical response suitable for applications in bone repair that support high loads (load-bearing).
  • Biodegradable bone grafts can be absorbed by the body and, after implantation surgery, their surgical removal is not necessary, for this reason, it has 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.
  • 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.
  • 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.
  • This topic is especially important for bone grafts with an open pore structure, whose bone growth process is carried out within the scaffold.
  • the mechanical properties are related to cell seeding, since mesenchymal stem cells (MSCs) differentiate if the stiffness is similar to the respective tissue.
  • MSCs mesenchymal stem cells
  • Calcium phosphates, calcium sulfates and bioactive glass are ceramic materials that have been used for bone regeneration. 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.
  • 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.
  • 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, glass 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), maintaining a porosity greater than 80%, this was due to the filling of micropores and microcracks on the surface by the polymer, preserving the interconnectivity of the bone graft or scaffold.
  • PLLA poly (D, L-lactic acid)
  • the Brazilian patent PI0300644B 1 deals with a process to prepare particulate biosilicates (from 0.1 and 30 microns), bioactive and resorbable (Biosilicate®) from glass or fritted plates, comprising the heat treatment of the material in one or two steps, milling and obtaining bioactive crystallized biosilicate powders of desired bioactivity, which in contact with body fluids produce a layer of HCA for restoration of teeth or can be gradually replaced by dental tissue and resorbed.
  • Biosilicate® bioactive and resorbable
  • the Brazilian patent PI1003676B2 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 made up of 50 to 98% of porogenic agent, from 2 to 50% of Biosilicate® and from 50 to 90% of liquids made up of 0.5- 15% binder and 85 to 99.5% liquid medium.
  • the binder, liquid medium and Biosilicate® are mixed, forming a suspension, which is ground, and the porogenic agent is added.
  • the suspension is dried into a powder, and the powder obtained is sieved, shaped into molds of any geometry, the shaped product is fired 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 pm.
  • Continuous glass fibers are obtained by downdrawing said composition, with lengths from millimeters to kilometers, and diameters between 2pm 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 Brazilian published application gives the glass high reactivity and low chemical durability, as is expected from a highly bioactive glass. Its greater vitreous stability allows it to be formed and/or undergo heat treatments, sintering, etc. without uncontrolled crystallization, thus allowing the use of simple processes, while requiring a low tendency to crystallization.
  • 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.
  • 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.
  • 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.
  • 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. Furthermore, it has high corrosion resistance and good biocompatibility.
  • an average cell size greater than 300 pm 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, these materials are synthesized using the replica method.
  • a sacrificial mold 60 ppi cylindrical polyurethane foams with 10 mm in diameter and 10 mm in height were used.
  • the pH of the suspensions was kept at 1.5 and under agitation for 2.5 hours at 5000 rpm.
  • a slip of titanium dioxide eg, 65 g of titanium dioxide in 25 mL of water
  • the porous mold is fired at a temperature of 400-550°C at a rate of 0.5°C/min for one hour.
  • 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.
  • 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.
  • the bone graft or scaffold can be subjected to centrifugation to remove excess slip.
  • sintering is carried out at 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.
  • 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 of 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).
  • the bone graft or scaffold can be centrifuged to remove excess slip.
  • the bone graft or scaffold is sintered at a temperature of 1500°C for 4 hours at a rate of 3°C/min.
  • a bone graft or titanium dioxide scaffold with three titanium dioxide coatings is obtained.
  • 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.
  • the third coating considerably increased the compressive strength of bone grafts or titanium dioxide scaffolds (3.39 ⁇ 0.77 MPa) compared to bone grafts or scaffolds that were coated only twice with titanium dioxide ( 1.78 ⁇ 0.52 MPa).
  • 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 (435-450pm).
  • the strategy used in the international patent document WO2014044666 Al 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 (1500 °C). This process allowed the internal porosity of the titanium dioxide struts to be partially filled, with an improvement in the densification of the material. It was found 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, minimum value for trabecular bone).
  • WO2014044666 Al 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. Furthermore, the vacuum facilitated the penetration of the slip into the microcracks and microporosities on the surface of the struts, allowing a considerable increase in the compressive strength. As a result, 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 the materials was that, after the second and third infiltration with titanium dioxide, centrifugation was performed to remove excess slip.
  • 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.
  • bone grafts or Biosilicate scaffolds were obtained using Biosilicate particles with an average size of 45 ⁇ m.
  • the bone grafts or Biosilicate scaffolds presented a very porous surface that facilitated the penetration of glass particles.
  • 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
  • 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.
  • 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.
  • the F18 bioglass forms a layer of HCA on the surface of the bone graft or Biosilicate scaffold, increasing the bioactivity of the biomaterial, which is very important for the intensification of 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 defunctionalization to induce the formation of a calcium phosphate layer on their surface.
  • Another important property of F18 glass and Biosilicate is that they are resorbable materials, avoiding a second surgery to remove them.
  • 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.
  • the US published application US20130330537A1 deals with a process for producing bone grafts by the method of coating a sacrificial mold with polyurethane (PU) 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 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 bone tissue regeneration.
  • 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 were initially the space occupied by the mold or PU foam.
  • the bone grafts or ceramic scaffolds obtained through the technique of replication 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.
  • 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 synthesizing 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.
  • 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:
  • Step I Preparation of the bone graft or Biosilicate® scaffold.
  • the bone graft or Biosilicate® scaffold object of patent PI0300644B 1.
  • a PU sponge with a medium cell opening in the range 35-55 ppi is used for the preparation of this bone graft or scaffold.
  • 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.
  • Step II The bone graft or Biosilicate® scaffold from Step I is vacuum infiltrated (-400 to -600mmHg) for 5 to 10 minutes with the same Biosilicate® slip from Etapal. The bone graft obtained in this step is dried at room temperature.
  • Step III the bone graft or Biosilicate® scaffold infiltrated in step II is submitted to firing, using the same procedure described in Step I.
  • 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.
  • Step V the bone graft or Biosilicate® scaffold that was infiltrated with the Bioglass F18 slip is submitted to firing under controlled conditions.
  • Step VI Steps IV and V are repeated.
  • Step VII The Biosilicate®/Bioglass F18 scaffold bone graft product with improved mechanical resistance to compression is recovered.
  • 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 the bone graft or scaffold.
  • 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.
  • 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 pm 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.
  • the 35 ppi polymeric sponge has a larger cell size, which reflects in the final result of compressive strength.
  • 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 take the same applicable as engineering material.
  • 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 pm and compressive strength of 3.3 ⁇ 0.3 MPa
  • these properties allow the bone graft or scaffold to mimic the structure of trabecular bone, facilitating the process of osteogenesis.
  • 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 resistance to the tissue during the healing process.
  • 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.
  • Another objective of the invention is a process of preparing a bone graft or scaffold of high bioactivity to facilitate the osteogenesis process.
  • Another objective of the invention is a process of 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).
  • 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 pm and 770 pm so as to achieve compressive strength between 1.00 ⁇ 0.2 MPa and 3.3 ⁇ 0.3 MPa.
  • 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 600°C, joining the microcracks in the Biosilicate® struts, reducing the size of the pores, but without them being clogged.
  • FIGURE 1 is an illustrative flowchart of the various steps of the bone graft or scaffold preparation process according to the invention.
  • 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 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.
  • FIGURE 3 illustrates the stress-strain curve for bone grafts or Biosilicate® scaffolds using 35 ppi PU foam.
  • Attached FIGURE 4 illustrates the stress-strain curve for BioS-2P/Bioglass F18 bone grafts or scaffolds using 35 ppi PU foam.
  • 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.
  • FIGURE 6 illustrates micrographs obtained by SEM showing the porous surface of the bone grafts or scaffolds of Biosilicate® obtained by infiltration in PU foam of 45 ppi 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.
  • FIGURE 7 illustrates micrographs (SEM) of bone grafts or scaffolds obtained from 45 ppi PU foams, under the following conditions:
  • FIGURE 7A shows SEM micrograph (50x amplification) with porous bone graft structure or highly interconnected scaffold after foam infiltration with Biosilicate® slip with 45 ⁇ m particle size;
  • 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 ⁇ m 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.
  • FIGURE 8 shows 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.
  • FIGURE 9 shows a bar graph comparing compressive strengths between BioS-2P/Bioglass F18 bone grafts or scaffolds synthesized using 45 and 35 ppi foam; and Biosilicate® bone grafts or scaffolds synthesized using 35 and 45 ppi foam.
  • 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
  • Biosilicate® bone graft or scaffold. It is a bone graft or scaffold prepared in accordance with the teachings of the Brazilian patent BR0306444B 1, of the Applicant and hereby fully incorporated by reference.
  • BioS-2P is a product of the Biosilicate® family and as such will be used in this report as the equivalent of Biosilicate®.
  • Bioglass F18 material prepared according to the teachings of Brazilian application BR102013020961, by the Applicant and fully incorporated herein by reference.
  • 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.
  • 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.
  • PU foams with 35 and 45 ppi were used.
  • 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.
  • 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 pm, these being suitable ranges for application of the product obtained as a trabecular implant.
  • 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 in (110), the preparation of the bone graft or scaffold of Biosilicate® is made according to the Brazilian patent PI0300644B 1.
  • a polyurethane sponge is used (PU) with average cell opening in the range 35-55 ppi (“pores per inch” or pores per inch).
  • 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.
  • Biosilicate® is used in the form of powder with an average particle size of 45 pm (particle size distribution range: 1-10Opm).
  • polyvinylbutyral PVB
  • PVA polyvinyl alcohol
  • CMC carboxymethyl cellulose
  • liquid media such as isopropyl alcohol and even water, such aspects being not critical for the present process.
  • 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.
  • the PU foam is dipped in the 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.
  • 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 consists of the following steps: a) Heating at a rate of 1-5°C/min until the burn-out temperature of the binder (PVB) and foam (PU), at 400-450°C; b) Level of 4 hours at a temperature of 350-450°C; c) Reheating at 1-5°C/min until the Biosilicate® sintering temperature (900-1100°C); d) Level of 3-5 hours at a temperature of 900-1,100°C; and e) Cooling to room temperature at a rate of 1 -5°C/min.
  • Biosilicate® occurs at high temperatures ( ⁇ 1000°C) 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.
  • the bone graft or scaffold of Biosilicate® (with mechanical resistance lower 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.
  • Step II in (120), the bone graft or Biosilicate® scaffold is infiltrated under vacuum (-400 to -000mmHg) with a Biosilicate® slip whose composition (Table 1) was described in Step I; however, a Biosilicate® powder with an average particle size of 5 pm is used (particle size distribution range: 0.1-20 pm).
  • 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 -OOOmmHg) 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.
  • Step III in (130), the bone graft or Biosilicate® scaffold that was infiltrated again with Biosilicate® slip is submitted to firing, using the same procedure described in Step I above in this report.
  • 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).
  • the particle size analyzer (Horiba-LA-93) is used in isopropyl alcohol P.A.
  • 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 pm (particle size distribution range: 0.1- 20 pm). In addition to Bioglass F18, powdered polyvinylbutyral (PVB) is used as a binder and anhydrous ethyl alcohol as a liquid medium.
  • PVB polyvinylbutyral
  • 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.
  • 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 l-5 °C/min until the binder (PVB) elimination ("hirn-oil") temperature, at 400-450°C; b) Level of 4 hours at a temperature of 400-450°C; c) Reheating at 1-5°C/min until the sintering temperature of Bioglass F18 (600-900°C); d) Level of 3-5 hours at a temperature of 600-900°C; and e) Cooling to room temperature at a rate of 1-5°C/min.
  • PVB binder
  • 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 of this contributes to increasing the final mechanical strength of the bone graft or scaffold.
  • Step VI in (160), Steps IV (140) and V (150) above are repeated in this report.
  • 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 pm.
  • 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 mechanical strength greater than 3 MPa, that is, compatible with bone grafts or scaffolds in the form of small blocks available on the market.
  • Bioglass F18 increases the bactericidal effect of the bone graft or scaffold.
  • Scanning Electron Microscopy 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.
  • the porosity was calculated through the geometric density.
  • the length, height and width of the prism on its four faces were measured, and for the cylindrical samples, the diameter and height were measured, and the mean value was calculated.
  • 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.
  • the average pore size was measured using the Image -J software.
  • Table 3 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.
  • 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.
  • the Biosilicate® scaffold was infiltrated with the Bioglass F18 slip, the vacuum allowed the slip was able 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 pm 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.
  • the process of the invention contemplates two measures: a) Using a coarse Biosilicate® powder (45 pm) in the first infiltration procedure to increase porosity on the surface of bone grafts or scaffolds, facilitating slip penetration in subsequent infiltrations; and b) Use very fine powders (5 pm) for the preparation of the slips used in the following infiltrations, facilitating the penetration of the slips into the hollow structure of the bone grafts or scaffolds.
  • the mean cell size for the PU foam (45 ppi) was 590 ⁇ 170 pm and for the bone grafts or BioS-2P/Bio glass F18 scaffolds it was 525 ⁇ 220 pm. Furthermore, particle size distributions were found in the range of 310-1230 pm and 230-1140 pm, respectively.
  • the total porosity of the bone grafts or BioS-2P/Bioglass F18 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%.
  • the average cell size of the 45 ppi foam (590 pm) and the average cell size of the BioS-2P/Bioglass F18 scaffolds (525 pm) 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.
  • the bioglass 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.
  • the glass penetrated the interior of the struts, partially filling the internal porosity of the structure.
  • a highly porous and interconnected structure with a cell size of 525 pm and porosity above 80% are essential for cell penetration and migration, tissue growth, angiogenesis and transport of nutrients, oxygen and waste.
  • the bone graft or scaffold obtained by the process of the invention from 45 ppi PU foam presents useful compressive strength 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.
  • 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.
  • the bone graft or scaffold obtained with 45 ppi foam has an average cell size smaller, in the adequate range so that, after infiltration with Bioglass F18 slip, it presents adequate compressive strength for the desired purpose.
  • 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.
  • Figure 9 is interesting, showing not only the results of Table 4, but also the same parameters measured for the bone graft or Biosilicate® scaffold without infiltration of Bioglass F18 slip.
  • Figure 9 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.
  • a possible strategy to increase the mechanical strength of bone grafts or scaffolds would be to use a 60 ppi foam with a smaller strut size.
  • the hollow part left by the sponge would be smaller.
  • a similar behavior was observed when the foam cell size decreased from 35 to 45 ppi. Therefore, it is expected that a foam with a smaller cell size (370pm) 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.
  • 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%.
  • MSCM Mesenchymal Stem Cell Culture Medium
  • the expansion was performed according to the kit manufacturer's recommendations.
  • the plates were incubated for 4 days at 37 °C with 5% COi in an incubator (ThermoScientific-Steri-Cycle) until reaching complete confluence.
  • the MSCM was changed every two days.
  • 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.
  • the cells were subcultured in a 24-well plate with a density of 1 x 10 5 cells. cm -2 , using 0.5 mL of MSCM per well, being incubated at 37°C with 5% CO2 for 4 days until 95-100% confluence. These cells were used as controls and others as samples named dosion 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.
  • the solution was removed and the hASCs were subcultured over the bone grafts or scaffolds at a density of 1 x 10 5 cells. cm -2 using 1 mL of MSCM, completely covering the bone grafts or scaffolds. Plates were incubated at 37°C with 5% CO2 for 4 days until 95-100% confluence. The MSCM was changed every two days. All samples and controls were performed in triplicate.
  • 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 group of bone grafts or scaffolds.
  • 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.
  • the medium was filtered with sterile syringe filter (VWR-0 0.2 ⁇ m) 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.
  • 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 (S ⁇ o Cario s/SP-Brasil ).
  • 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.
  • the cells of the ion group and the bone grafts or scaffolds were in the process of adaptation, since the osteogenic medium was saturated with the ionic dissolution products of the scaffolds. bone grafts or scaffolds, affecting enzyme production.
  • 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.
  • 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.
  • 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.
  • bone grafts or scaffolds promote osteogenic differentiation of stem cells derived from human adipose tissue. It is observed that bone grafts or scaffolds were able to induce the expression of different factors of the BMP superfamily, TGF superfamily receptor, transcription factors, integrin and collagen receptors, all essential for osteogenesis.

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Abstract

L'invention concerne un procédé de préparation de greffons osseux consistant : (110) à préparer un Biosilicate®, (120) à infiltrer une barbotine de Biosilicate® dans le Biosilicate® de l'étape (110), (130) à brûler le Biosilicate® infiltré de l'étape (120), (140) à infiltrer le Biosilicate® brûlé de l'étape (130) avec du bioverre F18, (150) à brûler le produit de l'étape (140), (160) à répéter l'infiltration avec du bioverre F18 de l'étape (140) et la combustion de l'étape (150), et (170) à récupérer le produit Biosilicate®/bioverre F18 avec une résistance à la compression améliorée. Les greffons osseux obtenus présentent une porosité de 83,0 ± 2%, une taille moyenne de cellule de 770 ± 290 μm et une résistance à la compression de 1,0 ± 0,2MPa à une porosité de 82,0 ± 1,3%, une taille moyenne de cellule de 525 ± 220 μm et une résistance à la compression 3,3 ± 0,3 MPa étant donné qu'ils sont préparés respectivement à partir de mousses de PU 35 ppi ou 45 ppi. Les greffons osseux sont utilisés comme substituants de l'os trabéculaire et permettent d'obtenir une intégrité mécanique consistante et une porosité pour la régénération de tissus osseux fonctionnels.
PCT/BR2021/050372 2020-08-31 2021-08-31 Procédé de préparation de greffons osseux et greffons osseux obtenus WO2022040771A1 (fr)

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BR102020017697-8A BR102020017697A2 (pt) 2020-08-31 2020-08-31 Processo de preparação de enxertos ósseos e enxertos ósseos obtidos

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Citations (7)

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BR0300644A (pt) * 2003-02-20 2004-11-16 Univ Fed De Sao Carlos Processo de preparação de biosilicatos particulados bioativos e reabsorvìveis, composições para preparar ditos biosilicatos, biosilicatos particulados bioativos e reabsorvìveis e uso dos mesmos no tratamento de afecções bucais
BRPI1003676A2 (pt) * 2010-04-06 2012-04-17 Fundacao Universidade Fed De Sao Carlos suspensões para preparação de enxertos ósseos (scaffolds) à base de biosilicato, enxertos ósseos obtidos e processos de obtenção dos mesmos
US8258117B2 (en) * 2000-06-29 2012-09-04 Piramal Healthcare (Canada) Ltd Composition and method for the repair and regeneration of cartilage and other tissues
BR102013020961A2 (pt) * 2013-08-12 2016-03-08 Univ Fed De São Carlos composição vítrea, fibras e tecidos vítreos bioativos e artigos
US10478528B2 (en) * 2013-03-14 2019-11-19 Prosidyan, Inc. Bone graft implants containing allograft
US20200164100A1 (en) * 2013-03-06 2020-05-28 3D-Matrix Ltd. Surgical methods employing purified amphiphilic peptide compositions
US10835642B2 (en) * 2014-11-13 2020-11-17 Bioventus, Llc Moldable bone graft compositions

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8258117B2 (en) * 2000-06-29 2012-09-04 Piramal Healthcare (Canada) Ltd Composition and method for the repair and regeneration of cartilage and other tissues
BR0300644A (pt) * 2003-02-20 2004-11-16 Univ Fed De Sao Carlos Processo de preparação de biosilicatos particulados bioativos e reabsorvìveis, composições para preparar ditos biosilicatos, biosilicatos particulados bioativos e reabsorvìveis e uso dos mesmos no tratamento de afecções bucais
BRPI1003676A2 (pt) * 2010-04-06 2012-04-17 Fundacao Universidade Fed De Sao Carlos suspensões para preparação de enxertos ósseos (scaffolds) à base de biosilicato, enxertos ósseos obtidos e processos de obtenção dos mesmos
US20200164100A1 (en) * 2013-03-06 2020-05-28 3D-Matrix Ltd. Surgical methods employing purified amphiphilic peptide compositions
US10478528B2 (en) * 2013-03-14 2019-11-19 Prosidyan, Inc. Bone graft implants containing allograft
BR102013020961A2 (pt) * 2013-08-12 2016-03-08 Univ Fed De São Carlos composição vítrea, fibras e tecidos vítreos bioativos e artigos
US10835642B2 (en) * 2014-11-13 2020-11-17 Bioventus, Llc Moldable bone graft compositions

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