ES2913574B2 - BIODEGRADABLE AND ANTIMICROBIAL SCAFFOLD FOR BONE TISSUE ENGINEERING AND PROCEDURE FOR OBTAINING IT - Google Patents

Description

DESCRIPTION

BIODEGRADABLE AND ANTIMICROBIAL SCAFFOLD FOR BONE TISSUE ENGINEERING AND PROCEDURE FOR OBTAINING IT

field of invention

The present invention is encompassed within the production of biodegradable scaffolds with application in bone tissue engineering in the treatment, regeneration and recovery of bone defects. Specifically, the present invention refers to a biodegradable scaffold with a porous structure with osteoinductive capacity and optimal mechanical and water absorption properties for use in bone tissue engineering, which also has antibacterial activity to combat infections at the bone defect site. regenerate, including those caused by multi-resistant pathogens to antibiotics. The present invention also includes a process for obtaining said scaffolds with direct application in bone tissue engineering and the use of these scaffolds as a component of implants, grafts and bone graft substitutes.

Background

A bone defect is a loss of bone in any part of the body where, under normal conditions, it would have to be present [1]. Bone defects can arise from congenital anomalies such as fibular hemimelia [2], or acquired anomalies such as fractures, diseases (osteoporosis and osteosarcomas), surgeries (surgical tumor removal) and infections [2-8]. A bone defect is critical when, having adequate biology, there is an inability to replace abundant bone loss and that, despite adequate stabilization, does not ossify spontaneously [5,7,9,10-13]. This generally occurs when the size of the defect is greater than 1-2 cm or when there is a loss of more than 50% of the bone circumference. In the same way, the biomechanical conditions, the age of the patient, the metabolic and systemic state of the same and the related lesions that affect the ossification of the defect are other conditions that influence its determination [7]. It must be emphasized that a critical bone defect cannot ossify adequately without the therapeutic aid or with materials designed to promote bone regeneration [5]. Taking into account all the factors listed above, the treatment method is not universal, but is selected individually for each patient. First of all, it is necessary to know the conditions that must occur for proper bone healing, in order to understand why critical bone defects do not follow proper healing. The healing of a bone, also called ossification, is similar to the healing of a tissue, with the inflammatory, proliferation and remodeling phases. Bone is a tissue that has the capacity for remodeling both at a functional and structural level [5]. The objective of the ossification of a bone is to reach a functional capacity and a histological structure that is as similar to the initial one. That is, regenerate the mineralized tissue at the lesion site and also restore mechanical resistance [14]. There are two types of ossifications, primary or direct ossification and secondary or indirect ossification. On the one hand, in primary ossification, bone is formed directly at the fracture site. For this, the two requirements that must be met are: having a maximum of 1 mm between the bone fragments and less than 2% of micromovements in the fracture site [15]. Secondary or indirect ossification, on the other hand, is characterized by the formation of connective tissue called callus, which evolves from fibrous to cartilaginous tissue, to finally generate bone tissue at the fracture site. This last state is followed by a remodeling phase that can continue for years until the original structure and resistance of the bone is achieved [5-7,10,14,15]. For these reasons, it is considered that critical bone defects do not follow primary ossification, but secondary ossification; but that require external support, since they cannot solve it by themselves [5].

Accordingly, various therapeutic methods exist to treat critical bone defects. To this end there are natural and synthetic therapeutic methods. On the one hand, within the natural therapeutic methods, there are: autografts (bone transplants from a donor area of the same individual, but far from the pathological region, to reconstruct a defect within the same patient [2,16]); allografts (grafts from cadavers or living donors of the same species, but genetically different[16,17]) and xenografts (these are grafts of natural origin, but they come from another species such as bovine species or derived from coral [14,17-19 ]). Within the autografts, there are cancellous bone and cortical bone. Autografts Cancellous grafts have the desired osteogenic and osteoinductive properties [2,6,10,11,16-18] and cortical autografts are mostly osteoconductive grafts with very little osteoinductive capacity but provide great mechanical support [18,19]. For these reasons, cortical autografts are optimal for critical bone defects as they require high structural mechanical support [18]. On the other hand, in contrast to natural therapeutic methods, there are synthetic ones to solve critical bone defects. Within these we can differentiate implants or prostheses and scaffolds. In the first place, the implants or prostheses replace the presence of natural material in the bone defect, but they are not intended for the body to take advantage of the implant to be able to ossify. Within traditional implants, the classic materials that have been used are: stainless steel, chrome and cobalt alloy, titanium or titanium alloy. But these materials cannot mechanically replicate bone, which may result in them not properly integrating into the recipient [4,10,16]. For this reason, there has been an evolution of these implants that replace tissue with synthetic biomaterials that are integrated into it. Currently, the main options for regenerative treatment of bone defects are autologous, allogeneic or synthetic implants, but none of these techniques has proven to be completely satisfactory [2,8,16]. These limitations have led scientists and clinicians to focus their research on aspects of tissue engineering and regenerative medicine such as bone reconstruction and on the development of supports, scaffolds and/or matrices, also called "scaffolds" to promote bone regeneration [2, 3,6,8] These scaffolds are composed of biomaterials and can be used for the treatment of bone defects.To do this, they must fulfill the following specific functions: act as a cell substrate, supply nutrition, administer bioactive molecules, and provide stimuli to direct cells to their destination [6].To perform such functions, these scaffolds must have the ideal characteristics of being osteogenic, biocompatible, biodegradable, capable of providing structural support, easy to implant, and cost-effective [1,6]. The materials with which the scaffolds are made are bioactive and resorbable over time. te and are replaced by host tissues, which facilitates repair in situ. For this, engineers must be inspired by the natural composition and structure of bone [16]. These biomaterials must not be mutagenic, antigenic, carcinogenic or teratogenic, nor toxic, they must also allow osseointegration without causing an adverse immunological reaction [4,6].

Various biomaterials can be chosen depending on the objective (mechanical resistance or filler) and the approach (percutaneous or surgical) that are selected from polymers, ceramics or mixtures thereof. Furthermore, some biomaterials are composed of growth factors such as bone morphogenic proteins (BMPs) to induce bone repair [16]. Ideally, biomaterials should be absorbable at a variable rate depending on their anticipated biomechanical function and should allow for the growth of newly formed blood vessels from neighboring tissues, with good-quality vascularization of the tissue in contact with the scaffold being crucial [10]. ,16].

Likewise, these scaffolds can be manufactured precisely, reproducing the anatomical characteristics of native bone, maintaining sufficient structural integrity during the growth and remodeling process in vitro and/or in vivo [8]. Similarly, proper scaffold construction must possess a highly porous three-dimensional network, with interconnected pores and surface properties, that are optimized for the attachment, migration, proliferation, and differentiation of the cell types of interest and that allow the transport of nutrients and metabolic wastes. In addition, they must possess a defined biocompatibility and biodegradability rate to complement tissue growth [8].

No less important than the previous requirements is the fact that the implantation of the scaffold in the area where the bone must be regenerated necessarily requires surgery, which, as is known, can be compromised by the easy appearance of bacterial infections. It is known that at least 10% of these interventions are associated with the appearance of bacterial infections, which compromise the efficacy and usefulness of the scaffold for the desired purposes.

Infections associated with implantable devices are characterized by bacterial adhesion, colonization, and development of a biofilm. The most common organisms associated with infections in implantable devices include the Gram-positive strains: Staphylococcus epidermis, Staphylococcus aureus, and Propionibacterium acnes, as well as the Gram-negative strains Pseudomonas aeruginosa. Osteomyelitis is inflammation of the bone and can appear as a consequence of biofilm formation, causing a substantial increase in bone resorption and reactive bone formation.

The biofilm is composed of substances secreted by bacteria, such as proteins, lipids, lipopolysaccharides and DNA, forming a matrix around the bacteria that provides protection against the action of antibiotics and immune defenses. The bacteria present in the biofilm also have a higher mutation rate, being able to present greater virulence when exposed to antibiotics and favoring the generation of antibiotic-resistant phenotypes.

However, the measures currently being considered to try to prevent the appearance of infections associated with the use of scaffolds are not entirely satisfactory. On the one hand, these are limited to the administration of high doses of antibiotics together with the implantation of the scaffold in the affected area or directly integrating the antibiotic dose into the scaffold itself. The administration of these high doses of antibiotics supposes a considerable increase in the possibilities of side effects caused by the antibiotic, such as allergic reactions, as well as in general the toxicity of the scaffold. Alternatively, the incorporation of different ions in the matrices and structures that make up the scaffold is described, however, these measures can also be toxic or the release of ions (osteoinductors and antimicrobials) does not occur at the adequate speed to achieve, at the same time, induce regeneration and prevent infection

Patent ES2393602 describes ordered mesoporous silica materials that include three different antibiotics in their pores: linezolid, rifampicin and vancomycin. Spanish patent ES2405774 describes a scaffold based on one or more polysaccharides and a crosslinking agent comprising antimicrobials selected from among: alkylparabens, such as methylparaben, ethylparaben, propylparaben and butylparaben, cresol, chlorocresol, hydroquinone, sodium benzoate, potassium benzoate , triclosan, chlorhexidine, rifampicin, minocycline, chlorhexidine, silver ion agents and silver-based compositions.

Japanese patent JP5882997 describes a bone graft comprising a solid inorganic component and a hydrogel containing silicon ions.

Optionally it can contain other ions, for example strontium, magnesium, potassium, cobalt copper, nickel, zinc, selenium, silver and fluorine.

The present invention proposes a solution to the previously identified technical problems through the definition of a scaffold , with application in bone tissue engineering for the treatment and recovery of bone defects, including critical ones, with osteoinductive capacity that also prevents infections. , including those caused by bacteria that are multiresistant to antibiotics. That is, the present invention proposes an effective technical solution to an existing need in the technical field of bone tissue engineering, providing a scaffold capable of regenerating a bone defect, including a critical bone defect, and at the same time treating any pathogenic infection, including multi-resistant infections that could compromise the bone regeneration process at the site of the bone defect to be treated.

Description of the invention

The present invention refers to a biodegradable scaffold with application in bone tissue engineering for the treatment, regeneration and recovery of bone defects in mammals, as well as a method for obtaining said medical devices.

The scaffold with application in bone tissue engineering according to the present invention is presented in the form of a three-dimensional (3D), porous, biodegradable and biocompatible structure and presents mechanical resistance to compression appropriate to the implant site. The structure that makes up the scaffold allows the development of new bone tissue in the bone defect to be recovered. Advantageously, the structure and components that make up the scaffold have an osteoinductive and antibacterial action during the bone regeneration process, being highly effective for the desired purposes.

For this reason, an object of the present invention is a scaffold for use in bone tissue engineering that comprises:

- a body, support or porous structure with a three-dimensional (3D) shape made from a polymeric material, and

- a hydrogel of calcium and zinc alginate,

characterized in that said calcium and zinc alginate hydrogel is integrated into the body, support or porous structure, and in that the calcium and zinc alginate hydrogel comprises between 4% to 7% by weight of zinc ions and 3% to 6 % by weight of calcium ions.

Another object of the present invention is a scaffold according to the previous paragraph, characterized in that the calcium and zinc alginate hydrogel integrated into the body, support or porous structure is arranged as a coating or filling of the structure or body. porous polymeric material.

Another object of the present invention is a scaffold according to the previous paragraphs, characterized in that the body, support or porous structure with a three-dimensional (3D) shape is made from a polymeric material selected from the group comprising: aliphatic polyesters, poly(amino acids), copoly(ether esters), polyalkylene oxalates, polyamides, polycarbonates derived from tyrosine, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amino groups, poly(anhydrides), polyphosphazenes, biomolecules, such such as collagen, elastin, hyaluronic acid, bioabsorbable starches, and mixtures thereof, aliphatic polyesters, such as, homopolymers and copolymers of lactide, including lactic acid, D-, L- and mesolactide, glycolide, including glycolic acid), ? -caprolactone, p-dioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate or, ?-valerolactone, ?-butyrolactone, ?-butyrolactone, ?-decalactone, hydroxybutyrate (repeating units), hydroxyvalerate (repeating units), 1,4-dioxepan-2-one, including its dimer 1 ,5,8,12-tetraoxacyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone, alpha , alphadiethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxane-2,5-dione, 3,3-diethyl-1,4-dioxane-2,5-dione, 6,8-dioxabicycloctan -7-one and polymer mixtures thereof.

Another object of the present invention is a scaffold according to the previous paragraphs, characterized in that the body, support or porous structure with a three-dimensional (3D) shape is made from polylactic acid (PLA).

It is also an object of the present invention a scaffold according to the previous paragraphs, characterized in that the calcium and zinc alginate coating comprises between 1.5 - 2% w/w (with respect to the total weight of the scaffold)

Also an object of the present invention is a scaffold according to the previous paragraphs, characterized in that the pore volume is 20 to 30% with an average pore size of 400 to 600 pm.

An object of the present invention is also a scaffold according to the previous paragraphs, characterized in that it optionally incorporates components selected from the group comprising: graphene, graphene oxide, carbon nanofibers, graphite, reduced graphene oxide, reduced graphene, nanotubes single or multi-wall carbon, fullerene, alkylamine- or ammonia-functionalized graphene oxide, boron-doped graphene, nitrogen-doped graphene, phosphorus-doped graphene, sulfur-doped graphene, boron-nitrogen-doped graphene, graphene-doped phosphorus and nitrogen, sulfur and nitrogen doped graphene and sulfonated reduced graphene oxide, nanocomposites with carbon nanomaterials such as graphene/TiO2, graphene/Fe3O4, graphene/Mn3O4, graphene/Pd, graphene/Pt, graphene/PtCo, graphene/ PtPd, reduced graphene/TiO2, reduced graphene/Fe3O4, reduced graphene/Mn3O4, reduced graphene/Pd, reduced graphene/Pt, reduced graphene/PtCo and reduced graphene/PtPd and nan or polypyrrole particles, nanoparticles and/or nanofibers such as silver or gold nanoparticles, nanocellulose, or ceramic nanoclays.

Another object of the present invention is a process for obtaining a scaffold according to the previous paragraphs, which comprises the following stages: a) 3D printing of a support, structure or polymeric porous body (1);

b) Vacuum immersion of the structure in an aqueous solution of sodium alginate (2,3);

c) Sonication (4);

d) Immersion of the sodium alginate polymer structure in an aqueous solution of calcium chloride (5,6);

e) Immersion of the calcium alginate polymer structure in an aqueous solution of zinc chloride (7.8)

f) Purification or washing of the scaffold (10); and

g) Oven drying (9).

It is also an object of the present invention a process according to the previous paragraph, characterized in that the 3D printing of the support, structure or porous body is carried out with the fused deposition modeling technique.

An object of the present invention is also a process according to the previous paragraphs, characterized in that the vacuum immersion of the structure in step b) is carried out in an aqueous solution of sodium alginate at a concentration of 2% w/ v.

An object of the present invention is also a process according to the previous paragraphs, characterized in that the immersion of the structure in stage d) is carried out in an aqueous solution of calcium chloride at a concentration of 0.5% w/v and the The immersion of the structure in step e) is carried out in an aqueous solution of zinc chloride at a concentration of 0.5% w/v.

Another object of the present invention is a scaffold as defined in the previous paragraphs for use in reconstructive processes for bone defects such as: segmental defects, defects caused by trauma, osteolytic tumors, gunshot wounds, defects caused by osteoarthritis, surgery, osteomyelitis, tumor surgery, structural bone defects, defects caused by osteoporosis, osteopenia and/or metabolic bone disease.

Finally, an object of the present invention is an implant, graft and bone graft substitute that comprises a scaffold as defined in the previous paragraphs and in general in the present invention.

figures

Figure 1 represents the bottom and elevation view of a scaffold of the invention.

Figure 2 represents a diagram of the manufacturing process of the scaffold of the invention.

Figure 3 illustrates a 10 mm cross section PLA scaffold associated to the osteosynthesis plate by means of both screws in caudal view (A) and in lateral view (B).

Figure 4 illustrates the surgical procedure in the animal model. Conventional craniolateral approach to the femur (A), separation of the vastus lateralis from the surface of the femur to expose the femoral shaft (B), marking the bone with the cutting saw, positioning the plate with the scaffold on one side to section the length Exact image of the scaffold (C), holding the plate with reduction forceps to prevent its movement (D), making a hole with the drill guide until the two cortical bones of the bone are drilled (E), fixing the implant to the femur ( F).

Figure 5 represents the results of the degree of ossification of the bone defect following the scale described by Stacy et al. [twenty]

Figure 6 illustrates the 3D reconstruction of the femoral scans. Femur corresponding to the UCV-1 group (A) and femur corresponding to the UCV-0 group (B).

Figure 7 illustrates the results of the agar diffusion antibacterial assay against the Gram-positive bacterium MRSA and against the Gram-negative P. aeruginosa.

Figure 8 represents the FTIR spectrum in the region of 4000-650 cm-1. PLA (a) and PLA scaffolds with alginate filler crosslinked with calcium and zinc cations (b). The PLA scaffold with cross-linked alginate filler shows the incorporation of OH groups typical of hydrogels with peak widths between 4000 and 3500 cm-1.

Figure 9 (a, b) show two images of HE histological sections at 4X, of the bone-scaffold transition zone, (example 2).

Detailed description of the invention

As a result of an intense research process, the inventors of the present invention have succeeded in designing a biodegradable and porous scaffold for use in bone tissue engineering that comprises:

- a body or porous structure with a three-dimensional (3D) shape made from a polymeric material, and

- a hydrogel of calcium and zinc alginate,

characterized in that said calcium and zinc alginate hydrogel is found covering or filling the structure or porous body of polymeric material.

3D structure polymeric material

The materials with which the scaffolds of the present invention are manufactured are bioactive and resorbable over time. They gradually degrade and are replaced by host tissues, facilitating bone defect repair in situ. The materials that make up the body or porous structure of the scaffold according to the present invention are non-mutagenic, antigenic, carcinogenic or teratogenic, nor toxic biomaterials that also allow osseointegration without causing an adverse immunological reaction.

Examples of suitable bioabsorbable biocompatible polymers that could be used include polymers selected from the group consisting of aliphatic polyesters, poly(amino acids), copoly(ether esters), polyalkylene oxalates, polyamides, tyrosine-derived polycarbonates, poly(iminocarbonates), polyorthoesters , polyoxaesters, polyamidoesters, amino-containing polyoxaesters, poly(anhydrides), polyphosphazenes, biomolecules (ie, biopolymers such as collagen, elastin, hyaluronic acid, bioabsorbable starches, etc.), and mixtures thereof. For the purpose of the invention, aliphatic polyesters include, but are not limited to, homopolymers and copolymers of lactide (including lactic acid, D-, L- and mesolactide), glycolide (including glycolic acid), ε-caprolactone, pdioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, δ-valerolactone, β-butyrolactone, γ-butyrolactone , ?-decalactone, hydroxybutyrate (repeating units), hydroxyvalerate (repeating units), 1,4-dioxepan-2-one (including its dimer 1,5,8,12-tetraoxacyclotetradecane-7,14-dione), 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone, alpha, alpha-diethylpropiolactone, carbonate ethylene, ethylene oxalate, 3-methyl-1,4-dioxan-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione, 6,8-dioxabicycloctan-7-one and polymer blends thereof.

Preferably, the use of polylactic acid (PLA) is contemplated as the only polymer to form the structure, body or porous support of the scaffold of the present invention. In this case, the use of commercial PLA filament for 3D printing from BQ (Madrid, Spain) is preferred for the fused deposition modeling (FDM) technique marketed in bovines with 1.75 mm filament and 1 kg weight. Preferably, the porous structure is made up of PLA with a ratio of 1.44±0.01 (% by weight of carbon/% by weight of oxygen) determined by elemental analysis with a field emission scanning electron microscope (Zeiss Ultra Model 55) at a voltage of 20KV, for example. Additionally, the use of other polymers is also contemplated, such as polycarbonate or polyvinyl alcohol for the formation of the structure or porous body.

alginate hydrogel

Alginic acid and its salts are polyelectrolyte biopolymers. It is made up of two types of monomeric units: beta-D-mannuronic acid and alpha-L-guluronic acid. Typically, alginic acid is shaped as a block polymer, with sections of mannuronic units alternating with sections of guluronic units. Alginate in the form of sodium, potassium or magnesium salt is soluble in water at pH greater than 3.5. On the contrary, alginic acid crosslinked with divalent cations such as calcium or zinc are insoluble in water. The presence of small amounts of divalent cations inhibits the hydration of alginates and high proportions of them cause their precipitation. For the purposes of the present invention, the scaffold comprises a three-dimensional (3D) shaped body or porous structure made from a polymeric material that is coated or filled with an alginate hydrogel crosslinked with calcium and zinc ions.

As will be detailed later, the alginate crosslinked with calcium and zinc ions is obtained once the body or porous structure has been coated with sodium alginate and it is crosslinked. The crosslinking of said sodium alginate occurs when the structure coated with sodium alginate is placed in contact with a calcium chloride solution and then with a zinc chloride solution, producing the replacement of sodium ions by calcium and zinc ions .

In this way, the alginate crosslinked with calcium and zinc that is part of the scaffold acquires its solid consistency as well as the desired stability of the scaffold.

For this reason, the sodium alginate selected for the present invention has an average molecular weight of 107.9 ± 2.7 KDa, and is made up of galuronic and mannuronic blocks but with the appropriate amount of galuronic groups (FG = 0.436 as a percentage of 1), which are the that crosslink in order to form a physically consistent gel but without having excessive crosslinking points that could retain too much zinc and therefore cause toxicity in the final product.

For the purposes of the present invention, it is also essential that the charge of calcium and zinc cations in the crosslinked alginate that forms part of the scaffold maintain certain proportions, since said balance turns out to be the key to the present invention in terms of providing physical consistency. to the alginate filler and through the zinc cations the biofunctionality (antimicrobial and regeneration inducer) in the optimal amount to avoid producing toxicity. According to the corresponding measurement carried out by means of elemental analysis in the field emission scanning electron microscope (Zeiss Ultra 55 Model) at a voltage of 20KV, it has been calculated that zinc is present in the alginate in the range of 4% to 7 % by weight, preferably 6.52% and 6.6% by weight and the presence of calcium ions in the range from 3% to 6% by weight, preferably 4.07 and 4.23% by weight. (The % by weight of zinc and calcium indicated corresponds to the percentage by weight of the atoms with respect to the rest of the present atoms, that is, C, H, O, Zn and Ca). Preferably, it is considered that the optimum charge ratio of both ions in the alginate is 1.58±0.02 (% by weight of zinc/% by weight of calcium).

Regarding the disposition of the calcium and zinc alginate in the body, structure or porous support of polymeric material, the alginate crosslinked with calcium and zinc is found covering said support and/or partially filling the porous cavities of the body, structure or support. That is, the incorporation of the crosslinked alginate in the structure maintains the porous structure of the scaffold, with a high degree of pore interconnectivity, necessary for it to exercise its osteoinductive capacity. That is, the open and interconnected porosity of the scaffold facilitates the colonization by cells, diffusion of essential nutrients and oxygen for cell survival, and waste products.

In an embodiment of the present invention, a scaffold whose porous structure is made of PLA has a calcium/zinc alginate coating between 1.5 and 2% weight/weight determined gravimetrically.

As shown in Figure 1, the porous support that makes up the scaffold of the present invention has a homogeneous pore structure, with a pore volume of 20% to 30% and an average size of between 400 and 600 pm. preferably 500 pm.

In an embodiment of the present invention, a scaffold whose porous structure is made of PLA has a porosity of 26.6% ± 0.05 by volume. When filled with the calcium/zinc alginate coating this porosity decreases slightly to 26.1%±0.05. These porosities were determined gravimetrically by filling the pores with distilled water and from the density measurements of the calcium/zinc alginate determined in the form of a film using the same sodium alginate and the same amount of calcium chloride and zinc chloride crosslinker. determined on a precision balance with an accessory for density by measuring the mass of the sample in air and immersed in n-octane at 25°C. Density (n=6) was: 2.022±0.012 g/cm3

From the water absorption by gravimetry and the measured density of the calcium/zinc alginate, the porosity of the scaffolds with and without calcium/zinc alginate coating was calculated.

Other optional components

The scaffold according to the present invention contemplates the additional incorporation of other components, although these components are not necessary for the scaffold to exert its osteoinductive and antibacterial action. These components can enhance and facilitate the desired actions on the scaffold and can be selected from between: nanomaterials that increase the electrical conductivity of the scaffold, such as conductive carbon nanomaterials (graphene, graphene oxide, carbon nanofibers, graphite, reduced graphene oxide, reduced graphene, single and multi-walled carbon nanotubes, fullerene, alkylamine or ammonia functionalized graphene oxide, boron-doped graphene, nitrogen-doped graphene, phosphorus-doped graphene, sulfur-doped graphene, boron-nitrogen doped graphene, phosphorus-nitrogen doped graphene, sulfur-nitrogen doped graphene and sulfonated reduced graphene oxide), nanocomposites with carbon nanomaterials such as graphene/TiO2, graphene/Fe3O4, graphene/Mn3O4, graphene/Pd, graphene/Pt, graphene/PtCo, graphene/PtPd, reduced graphene/TiO2, reduced graphene /Fe3O4, reduced graphene/Mn3O4, reduced graphene/Pd, reduced graphene/Pt, reduced graphene/PtCo and reduced graphene/PtPd)) and polypyrrole nanoparticles, nanoparticles and/or nanofibers as l Silver and gold nanoparticles, nanocellulose, nanoclays, ceramics, etc. The incorporation of these components is done through the alginate filler.

One of the most appreciable advantages of the scaffolds defined in the present invention is that the incorporation of zinc into the scaffold through the alginate provides an osteoinductive function as well as an antibacterial one, which makes it possible to avoid having to necessarily incorporate risk factors into the scaffold structure. growth as osteogenic factors, such as bone morphogenetic protein 2 (BMP-2), which can cause problems associated with immunogenicity, cancer risk, and alterations in cellular homeostasis. In addition to all this, there is a saving in costs and ease of the manufacturing process of the scaffolding.

An additional advantage of the scaffold defined in the present invention is that it does not require the incorporation of cells to promote bone formation, therefore it also results in cost savings and ease of the manufacturing process thereof.

scaffold fabrication method

For the manufacture of the body or porous structure with a three-dimensional shape of the scaffold, different methods and techniques are contemplated, among which are included: gas foaming, injection mold techniques from the melt, extrusion, electrospinning or electrospinning and preferably, prototyping techniques. fast. These techniques of Rapid prototyping includes the manufacture of structures layer by layer in a controlled process through certain software that previously defines the design. Rapid prototyping technologies include: fused deposition modeling ( FDM), stereolithography ( SLA), 3D bioextrusion printing ( 3D-P), cut and laminate manufacturing ( LOM) , and UV light curing ( SGC). .

Preferably, the present invention contemplates the use of the technique by fused deposition (FDM). This technique is based on the deposition on a platform of layers of molten material from the heating and softening of polymer filaments for the manufacture of the moulding. At the same time other soft filaments form support for the freely suspended surfaces of the moulding, in order to be able to be built. The FDM machine has a platform covered by a dense and flexible foam that moves vertically (z-axis) and a head equipped with two extruder nozzles for the heated filament: one for feeding the layers of the designed model and another for the construction of the supports. The filaments are stored in the machine, which is vacuum heated. The extrusion nozzles are fed through two rotating guides connected to a motor that transport the filaments stored in a roll. The software used by the FDM team is a combination of CAD/CAM not integrated into the machine, which connects to the computer with the CAM system, which is responsible for monitoring construction orders.

Once the body or 3D structure of the appropriate materials has been obtained, sodium alginate is integrated into said structure, which will later be crosslinked with calcium chloride and zinc chloride to give rise to the scaffold of the present invention.

This part of the manufacturing process, as shown in Figure 2, schematically comprises the following stages:

which comprises the following stages:

a) 3D printing of a support, structure or polymeric porous body (1);

b) Vacuum immersion of the structure in an aqueous solution of sodium alginate (2,3);

c) Sonication (4);

d) Immersion of the sodium alginate polymer structure in an aqueous solution of calcium chloride (5,6);

e) Immersion of the calcium alginate polymer structure in an aqueous solution of zinc chloride (7.8)

f) Purification or washing of the scaffold (10); and

g) Oven drying (9).

Specifically, once the porous support has been obtained, the process comprises vacuum filling the structure or porous body with sodium alginate and subsequently cross-linking with calcium chloride and zinc chloride. In this manufacturing process, the amount of calcium and zinc are key to the successful biofunctionality of the support: antimicrobial character and bone regeneration capacity without producing toxicity. Zinc is a very toxic compound that cross-linked as zinc chloride must remain in a minimum amount that allows the scaffold to be provided with antimicrobial and regenerative capacity without producing toxic effects. This is achieved by crosslinking the support initially by immersion in a low concentrated aqueous calcium chloride solution, 0.2-0.7% w/v, preferably 0.5% w/v, for 7-15 minutes, preferably 10 minutes. In this way, it is initially possible to bind a large amount of negative charges present in the polymer chains of sodium alginate with the divalent cations of calcium and achieve a solid consistency of the filling. Next, cross-linking with zinc chloride is carried out to load the alginate with a minimum amount that provides the support with the desired biofunctionality, antimicrobial and bone regeneration capacity, without producing toxicity. For this, a low concentrated aqueous zinc chloride solution is used, 0.25-0.5% w/v, preferably 0.5% w/v for 7-15 minutes, preferably 10 minutes. Subsequently, the supports are purified by washing each scaffold at least 3 times, for example, in 200 mL of distilled water for 10 - 20 minutes, preferably 15 minutes each time with magnetic stirring at a temperature between 20 - 35°C, preferably 25°C. Subsequently, the scaffolds are dried for 24-48 hours in an oven below the degradation of the scaffolds, preferably at a temperature between 40-70°C, preferably at 60°C.

This developed method has the great advantage over any other type of process in that, by means of a 3D scanner, it is possible to determine the exact dimensions of a bone defect generated by accident or disease and thus be able to produce custom 3D pieces with the dimensions and shape required in each case for each patient. The synthesis process also allows the additional incorporation of conductive nanomaterials such as conductive carbon nanomaterials (graphene, graphene oxide, carbon nanofibers, graphite, reduced graphene oxide, reduced graphene, single or multi-walled carbon nanotubes, fullerene, alkylamine-functionalized or ammonia-functionalized graphene oxide, boron-doped graphene, nitrogen-doped graphene, phosphorus-doped graphene, sulfur-doped graphene, boron-nitrogen-doped graphene, phosphorus-nitrogen-doped graphene, sulfur-nitrogen-doped graphene and sulfonated reduced graphene oxide), nanocomposites with carbon nanomaterials such as graphene/TiO2, graphene/Fe3O4, graphene/Mn3O4, graphene/Pd, graphene/Pt, graphene/PtCo, graphene/PtPd, reduced graphene/TiO2, reduced graphene /Fe3O4, reduced graphene/Mn3O4, reduced graphene/Pd, reduced graphene/Pt, reduced graphene/PtCo and reduced graphene/PtPd)) and polypyrrole nanoparticles, etc., through the re filled with alginate. By introducing conductive nanomaterials, it is possible to increase the electrical conductivity in the support that can be used to increase the regenerative and antimicrobial capacity when applying electrical stimulation. The synthesis process also allows the additional introduction of other types of nanomaterials, nanoparticles and/or nanofibers such as silver and gold nanoparticles, nanocellulose, nanoclays, ceramics, etc.

3D printing therefore makes it easier to obtain personalized scaffolds with specific shapes and dimensions for the bone lesion to be reconstructed, including critical bone defects.

By way of example, dimensions can range from 1. to 40. cm, and shapes vary from flat to cylindrical or to the desired shape depending on the bone application.

use of scaffolding

The scaffold object of the present invention finds application in the reconstruction of bone defects or lesions produced by different causes, whether they are those called segmental defects, produced by trauma, osteolytic tumors, firearm wounds, for example, or those caused as a consequence of osteoarthritis processes, osteomyelitis surgery or even tumor surgery. Also The application of the scaffolds according to the present invention is foreseen for the regeneration of structural bone defects, the cause of which could be osteoporosis, osteopenia or metabolic bone disease. In particular, the scaffolds according to the present invention are especially intended for processes of bone reconstruction of critical bone defects, which are defined as those defects where the organism fails to promote tissue repair of bone tissue, only forming scar tissue, there is an inability to replace extensive bone loss and it does not ossify spontaneously. This generally occurs when the size of the defect is greater than 1 - 2 cm or when there is a loss of more than 50% of the circumference of the bone.

regenerative and antibacterial activity

The scaffold of the present invention exerts osteoinductive as well as antibacterial activity against infections caused by specifically Gram-positive bacteria, such as Staphylococcus aureus. The scaffold of the present invention has been shown to have a great antibacterial capacity even against the multiresistant bacterium Staphylococcus epidermidis resistant to methicillin.

In conclusion, the present invention presents an antimicrobial biodegradable porous scaffold that is easy, cheap and reproducible to manufacture with great antimicrobial capacity, even against multi-resistant pathogens, and with adequate bone regeneration capacity and mechanical and water absorption properties, its manufacturing process and its application in bone tissue engineering. The porous support was initially prepared by 3D printing using the polylactic acid (PLA) Fuse Deposition Modeling (FDM) technique, resulting in a highly reproducible, automated preparation method that allows specific design for each bone defect with great precision ( 3d print). In addition, the manufacturing process is in accordance with the principles of green chemistry and sustainable development using water as a solvent and using minimal electrical energy in the preparation. The selected materials (polylactic acid and alginate) are biomaterials approved by the US FDA for biomedical use in humans. The filling material (alginate) is of natural origin and therefore renewable. Both polymers are biodegradable and therefore are expected to biodegrade. within the human body as the bone regenerates. They are low-priced polymers compared to others on the market. In addition, the invention has the great advantage of producing an antimicrobial scaffold that prevents infections, including multiresistant infections, as has been verified by the research group in vitro and in vivo tests. The invention also provides the possibility of incorporating nanomaterials of all kinds through vacuum filling of the alginate phase. Therefore, the material called UCV-1 presents great advantages with respect to all produced up to now for the regeneration of large bone defects. The invention can also be used for other applications such as the regeneration of other types of tissues by designing scaffolds with the morphology required for each tissue: neuronal, muscular regeneration, etc.; performing another type of design by 3D printing: reinforced antibacterial dressings for wound care; as biodegradable materials for antibacterial capacity for controlled drug delivery; manufacture of antibacterial biodegradable catheters; Hydrogel materials for immobilization of cells or enzymes in bioprocessing, eliminating the usual big problem of bacterial contamination.

Examples:

Example 1:

1.1. Design and manufacture of scaffolding

Polylactic acid (PLA) scaffolds with and without alginate filler crosslinked with calcium and zinc cations were fabricated using 3D printing technology by the Biomaterials and Bioengineering Research Group of the San Alberto Magno Translational Research Center of the University of Catholic of Valencia San Vicente Mártir (UCV). Therefore, scaffolds made of polylactic acid material will hereinafter be referred to as UCV-0 scaffolds and scaffolds according to the present invention made from PLA with crosslinked alginate filler will be hereinafter referred to as UCV scaffolds. -1. Scaffolds have been developed for this study with a cross section of 10 mm in diameter and length of 20 mm (Figure 1). For both designs, 4 scaffold replicas for the UCV-0 group and 4 replicas for the UCV-1 group were manufactured by 3D printing.

The structure, body or porous support of polylactic acid (PLA) has been initially prepared by 3D printing using the fused deposition modeling (FDM) technique. Figure 1 presents an image of the views of the PLA scaffold designed for 3D printing.

Subsequently, the porous support has been filled under vacuum with sodium alginate that was later crosslinked with calcium chloride and zinc chloride. The filling is carried out by immersing the support in the different solutions. The scheme of the scaffold manufacturing process can be seen in the following scheme of Figure 2.

In this manufacturing process, the amount of calcium and zinc are key to the successful biofunctionality of the support: antimicrobial character and bone regeneration capacity without producing toxicity. This is achieved by cross-linking the support initially by immersion in a slightly concentrated aqueous calcium chloride solution (optimum range of calcium chloride concentration determined experimentally 0.5% w/v) for 10 minutes and later also by immersion in an aqueous calcium chloride solution. of low concentrated zinc (suitable range of concentration of zinc chloride determined experimentally 0.25 - 0.5% w/v) for 10 minutes. In this way, it is initially possible to bind a large amount of negative charges present in the polymer chains of sodium alginate with the divalent cations of calcium and achieve a solid consistency of the filling. Subsequently, the supports are purified by washing each scaffold 3 times in 200 mL of distilled water for 15 minutes each time with magnetic stirring at 25°C. Finally, the scaffolds are dried for 48 hours in an oven below the degradation of the scaffolds, preferably at 60°C.

1.2. In vitro assembly of the stabilization system

In vitro testing was performed on each of the two designs, in which the scaffold was fixed to a 10-hole 2.7mm titanium locking plate (PAX®, Securos Surgical®, United States) with two 1/4-inch screws. 2.7 mm diameter by 12 mm length titanium (PAX®, Securos Surgical®, United States) (Figure 3).

Once the correct arrangement of the implants had been reviewed, they were sterilized using the ASP STERRAD® NX® system (United States), a low-temperature sterilization system with plasma gas. Each implant was composed for a scaffold, 2 screws and a locked plate, for which 8 implants were used in total; 4 for the UCV-0 group and 4 for the UCV-1 group.

1.3. Design, choice and realization of the model of the critical bone defect

In the present study, a 20-mm full-section segmental bone defect model was chosen in the mid-femoral shaft. To create this bone defect, a complete ostectomy of 20 mm was performed in vivo in the mid-femoral diaphysis of the left posterior extremity of the individuals of the animal model used in the study, ensuring that said location allowed the correct application of the implants.

1.4. animal model used

To carry out the pilot study, 8 New Zealand lagomorphs (Oryctolagus cuniculus) individuals, 1-year-old females, have been used. These individuals were randomly distributed into 2 treatment groups, the UCV-0 group and the UCV-1 group. Each group consisted of 4 individuals. Total estimated study time was 12 weeks (2 weeks of quarantine 10 weeks of post-surgical evolution). This experimental study was approved both by the Ethics Committee of the La Fe Research Institute, as well as by its Authorized Body; obtaining authorization from the Ministry of Agriculture, Livestock and Fisheries of the Valencia Community, in accordance with the provisions of article 31 of Royal Decree RD53/2013.

1.5. Pre-surgical management

First, the individuals remained in a 14-day quarantine period in the animal facility of the La Fe Research Institute before the surgical procedure. After 14 days of quarantine, the surgical intervention was carried out in which the critical bone defect was created for the subsequent placement of the scaffold with the stabilization system.

During pre-anesthetic management, a complete physical examination was performed in which the individuals were weighed. After weighing, the pre-medication for each individual was calculated. The pre-medication consisted of Medetomidine (50 pg/kg, Domtor® 1mg/ml solution for injection, Ecuphar, Finland), Fentanyl (10 pg/kg, Fentanest® 0.05 mg/ml solution for injection, Kern pharma, Spain), Ketamine (5 mg/kg, Imalgene® 100 mg/ml solution for injection, Merial, Spain) and Midazolam (0.5 mg/kg, Midazolam Normon® 15 mg/3 ml solution for injection, Normon Laboratories, Spain). A combination of the above was administered intramuscularly in the same syringe. Once the individual was under sedation, the marginal ear vein was catheterized, and the left posterior extremity was shaved, as well as washed and aseptically prepared. Subsequently, the individuals were induced by intravenous administration of Propofol (2 mg/kg, Propovet Multidosis® 10 mg/ml injectable emulsion, Ecuphar, Spain) for, after endotracheal intubation, maintenance with inhaled isoflurane. (Isofluteck® 1000 mg/g, Karizoo, Spain) following the CAM referenced for this species; 3.7 /- 0.16% 29 inhalation route. Fentanyl (0.01 mg/kg, Fentanest® 0.05 mg/ml injectable solution, Kern pharma, Spain) was administered as intra-surgical analgesia by continuous intravenous infusion. In the same way, the antibiotic Enrofloxacin (10 mg/kg, Baytril® 50 mg/ml solution for injection, Bayer, Spain) was administered intravenously, also during the intraoperative period.

1.6. Surgical procedure in the animal model

The individual was placed in right lateral decubitus once the limb was aseptically prepared. The patient was covered with surgical drapes, leaving the left posterior extremity free.

Regarding the approach, a craniolateral approach to the femur was performed in a conventional manner, as described by Piermattei (2014) (Figure 4A). Following the skin incision, an incision was made in the superficial lamina of the fascia lata along the cranial border of the biceps femoris muscle for the length of the incision. The biceps femoris was retracted caudally to expose the vastus lateralis muscle. An incision was made in the fascial septum of the vastus lateralis at its insertion on the caudal lateral border of the femur. The vastus lateralis was removed from the surface of the femur to expose the femoral shaft (Figure 4B).

Regarding the creation of the critical bone defect, once the femoral shaft was exposed, an ostectomy of the mid-femoral shaft of 20 mm in length was performed, thus creating the critical bone defect. To perform the ostectomy correctly, the bone was first marked with the cutting saw, positioning the plate with the scaffold on one side to section the exact length of the scaffolding. (Figure 4C). After the bone segment was removed, both ends of the femoral bone were fixed with reduction forceps to maintain the anatomical position and the scaffold was positioned in the critical bone defect created.

Regarding the reduction of the fracture with a scaffold, once it was correctly positioned, the plate was reduced with reduction forceps to prevent its movement (Figure 4D). Subsequently, the hole in the plate was drilled with the drill guide until both cortices of the bone were drilled (Figure 4E).

Regarding the fixation and stabilization of the fracture, the titanium plate was fixed to the two femur fragments with two screws placed in the most distal holes of both ends of the plate (Figure 4F).

Finally, regarding the closure of the approach, after washing the area with physiological saline solution, the approach was closed in layers with a 4/0 glyconate suture (Monosyn®, B. Braun, Germany) in a continuous pattern. simple of apposition

1.7. Post-surgical management in the animal model

Once the intervention was completed, metoclopramide (0.5mg/kg, Primperan® 10mg/2ml solution for injection, Sanofi, Spain) and Ranitidine (2mg/kg, Ranitidine Normon® 10mg/ml solution for injection) were administered intravenously. , Normon Laboratories, Spain); and subcutaneously Meloxicam (0.4 mg/kg, Metacam® 2 mg/ml solution for injection, Boehringer, Germany). Subsequently, the patients returned to the stable room of the animal facility. Each individual remained in an individualized box with movement restriction for 10 weeks.

Finally, the individuals were sacrificed 10 weeks after the surgical procedure. Once the individual was under sedation, following the previously described protocol, the marginal ear vein was catheterized, to subsequently administer an overdose of commercial sodium pentobarbital solution (Dolethal® 200 mg/ml injectable solution, Vetoquinol, Spain).

1.8. Functional evaluation of the scaffold: tomographic evaluation

After the sacrifice of the individuals, a tomographic study was performed with a 16-slice scanner (Siemens Somatom Scope® 16, Germany). For this, individuals They were positioned in supine position and scanned with settings of 130 kVp, 170 mAs, in soft tissue and bone algorithm, with a slice thickness of 1 mm.

For image analysis, commercially available DICOM image viewing software (Aycan Workstation v3.6) was used using a soft tissue window (width: 350 HU; level: 40 HU) and a bone window (width: 2000 HU, level: 600 HU).

The evaluation of the degree of ossification of the bone defects was carried out in two independent ways. The first mode consists of the quantitative assessment by measuring the Hounsfields Units (HU) and for this, 6 independent points were selected, in mid-cortical and medullary location at the proximal, mid and distal level of the bone defect. The second mode consisted of the semiquantitative assessment of the degree of ossification of the bone defect, according to the scale described by Stacy et al. (2014). [twenty].

1.8. Physicochemical characterization of supports

The porous supports UCV-0 and UCV-1 have been characterized by Fourier transform infrared spectroscopy (FTIR), water absorption, mechanical compression tests in the dry state and in the swollen state after immersion in water for 24 hours at 37°. C (human body temperature).

1.9. Antibacterial characterization of supports

The antibacterial properties of the supports have been characterized against a Gram-positive bacterium MRSA and a Gram-negative Pseudomonas aeruginosa by means of the agar diffusion method.

2. Results

2.1. Evaluation of the experimental model

The procedure for creating the critical bone defect in the femur, as well as the placement of the scaffold in said anatomical location, was carried out satisfactorily in all individuals, with no significant complications during the surgical procedure. One of the individuals in the UCV-0 group died one hour after the surgical procedure, due to causes unrelated to the experimental model or the object of study. Likewise, one individual from the UCV-1 group was sacrificed two weeks after surgery due to an infection at the level of the surgical focus, and two others as well. of the CTR group (group treated with UCV-0) at four weeks due to fracture collapse, which resulted in loss of alignment and stabilization of the fracture. For these reasons, a total of 4 individuals arrived at the final period of the study, 1 belonging to the CTR group (25% of the individuals operated on and recovered from the anesthetic procedure) and 3 belonging to the OTT group (group treated with UCV-1) ( 100% of the individuals operated on and recovered from the anesthetic procedure) (Table 1).

Table 1. Follow-up of individuals

2.2. Functional evaluation of the scaffold

2.2.1. tomographic evaluation

As previously mentioned, the evaluation of the degree of ossification of bone defects was carried out in two independent ways: a quantitative evaluation by measuring the HU, and a semi-quantitative evaluation according to the scale described by Stacy et al. (2014) [20]. Below, Table 2 shows the results in HU for each of the evaluated regions, as well as the mean of each one of them for cortical (peripheral) and medullary (central) areas.

.

individuals.

In summary, we can observe how the general average in the OTT group was 578.67 HU in the middle cortical region, and 176.56 HU in the medullary region, compared to 214.33 HU and 59 HU respectively in the CTR group.

For the semi-quantitative assessment, the degree of ossification of the critical bone defect generated in the individuals was assessed using the scale described by Stacy et al. (2014) [20]. As seen in Table 3, the CTR-1 individual obtained a score of 3 on the scale. In contrast, the individuals in the TTO group, individual TTO-1, TTO-2, and TTO-3, obtained a score of 4, 2, and 4, respectively.

Table 3 . Results of the degree of ossification of the bone defect following the scale described by Stacy et al. (2014) [20].

Where, according to Stacy et al. (2014) [20], score 2 indicates signs suggestive of minimal or early ossification with slight mineralization between bone fragments without contact, a fine bridge connecting both bone fragments, or close contact between both fragments with slerotic ostectomy margins. Score 3 indicates mild fixation with bridging along less than 50% of the anteroposterior dimension of both fragments, with the bone fragments displaced or aligned in the anteroposterior dimension. Score 4 indicates moderate fixation with bridging along 50% or more of the dimension anteroposterior of both fragments, with the bone fragments displaced or aligned in the anteroposterior dimension with visible remnants of the previous ostectomy. In the same way, the results obtained from the degree of ossification of the bone defect are also observed in a graph following the scale described by Stacy et al. (2014) [20] (Figure 5).

Figure 6 shows a tomographic comparison of the ossification of the bone fragments between the UCV-0 control group and the UCV-1 group. This figure shows a more advanced degree of callus maturation in the UCV-1 group than in the UCV-0 group.

2.2.2. Antibacterial characterization of scaffolds

The UCV-1 scaffolds showed antibacterial capacity against the multiresistant Gram-positive bacterium Staphylococcus aureus (MRSA) and against the Gram-negative Pseudomonas aeruginosa (see Figure 7).

As shown in Figure 7, it can be seen how the UCV-1 scaffold produces a very clear inhibitory halo around the Gram-positive MRSA bacterium and not as potent but clearly observable against the Gram-negative bacterium, unlike of the UCV-0 scaffold that does not inhibit bacterial growth.

2.2.3. Physicochemical characterization of the scaffolds

The FTIR spectroscopy results confirm the synthesis of the crosslinked alginate filler on the PLA support, since in Figure 8(b) a broad peak (between 4000 and 3500 cm-1) appears in the characteristic area of the OH hydrophilic groups. of the alginate compared to its absence in the unfilled PLA support, Figure 8(a).

In addition, as shown in Table 4, the immersion absorption result shows at human body temperature (37°C) that the UCV-1 scaffold absorbs water faster and in greater quantity when containing the crosslinked alginate filler.

Table 4 . Equilibrium water absorption (h= (dry mass-swollen mass/dry mass) in immersion at 37°C reached in both types of scaffolds from the dry state (zero time) up to 100 days.

The compressive mechanical properties in the dry and swollen state at 37°C (human body temperature), which are critical for bone regeneration applications, are also improved by the incorporation of the crosslinked alginate filler with both calcium and zinc cations (Table 5).

Table 5 . Mechanical compression tests of the UCV-0 (control) and UCV-1 scaffolds in the swollen state for 24 hours at 37°C and in the dry state.

Thus, the alginate filler in both the dry and swollen state within the scaffold provides physical integrity to the PLA scaffold by improving its compressive mechanical properties.

In conclusion, a porous scaffold capable of regenerating bone has been developed, preventing infections, including multiresistant infections that are currently causing so many problems and the World Health Organization estimates that by 2050 it will be the leading cause of death, surpassing cancer. It is a very reproducible and very economical process. In addition, it allows the production of scaffolds on demand for each patient according to the needs, performing a previous scan of the patient's bone defect. 42% of the individuals were sacrificed due to infection problems at the fracture site; being 100% of them belonging to the UCV-0 group. However, it is concluded that through the tomographic evaluation, especially in the quantitative assessment, the formation of more mature callus can be observed in the UCV-1 group than in the UCV-0 group, suggesting a more advanced ossification in the UCV- group. 1 of scaffolds made with porous PLA by 3D printing filled with crosslinked alginate with divalent cations of calcium and zinc in the appropriate proportion. In addition, these supports proved to have greater water absorption capacity and better mechanical behavior under compression in the dry state and swollen at human body temperature. The success of the physical consistency in remaining the cross-linked alginate filler within the scaffold was verified by FTIR infrared spectroscopy.

Example 2: Histological test

1. Methodology

1.1. Sampling

After the sacrifice of the individuals, the histological samples were extracted, for which the region of the femoral sample under study (middle diaphysis of the femur) was exposed, and by using a number 11 scalpel blade and a With an oscillating saw, the region of the scaffold with its bone callus was extracted, taking a margin of 1 cm proximal and distal to it.

1.2. histological study

The femoral portion that includes the scaffold is fixed in 4% paraformaldehyde at room temperature for at least 24h, for subsequent decalcification in a commercial EDTA solution (Osteodec®, Bio-Optica; Italy) for 21 days (until the correct decalcification).

After this period, the samples were included in paraffin blocks for sectioning and subsequent conservation. From each sample, 3 5-micrometer-thick sagittal sections were obtained with a microtome for subsequent staining with Hematoxylin-Eosin (H-E). The histological sections were firstly hydrated and deparaffinized using the usual protocol of immersions in Xylol and alcohols.

1.3. microscopic evaluation

The microscopic evaluation was carried out with a high-resolution optical microscope equipped with different objectives (4X, 10X, 20X, 40X and 60X) (Olympus)

The sections stained with H-E were subjected to evaluation following the modified semi-quantitative scale described for histological evaluation of critical bone defects (Oztürk et al., 2006) (Figure 9 a+b).

1.4. Results.

Table 6 is shown below, where the histological evaluations for each of the samples are expressed.

Table 6 : Values obtained in the histological evaluation for each individual: control sample of polylactic acid material (UCV-0) and the three replicates of the scaffolds made from PLA with calcium and zinc crosslinked alginate filler (UCV-1). )

As illustrated in Figure 9, in the histological samples a transition region is observed in the UCV-1 individual (Replica 1) formed by structured fibrocartilaginous tissue that is introduced through the pores of the scaffold achieving an adequate integration of the same with fibrous callus. In other sections of the same individual (not shown in the current image), more advanced callus stages are found in peripheral regions with cartilage tissue present. On the contrary, the UCV-0 sample shows a transition region formed by a destructured fibrous tissue, which fails to enter the pores of the scaffold, not achieving its expected integration.

In conclusion, the UCV-1 group seems to show a fibrocartilaginous callus in some of the studied regions, more advanced than that observed in the UCV-0 individual. The integration of the scaffold would be greater in the UCV-1 group, by allowing the formation of fibrous callus in its trabeculate, as well as in its peripheral region.

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Claims (13)

1. - A scaffold for use in bone tissue engineering comprising: - a body, support or porous structure with a three-dimensional (3D) shape made from a polymeric material, and - a hydrogel of calcium and zinc alginate, characterized in that said calcium and zinc alginate hydrogel is integrated into the body, support or porous structure, and in that the calcium and zinc alginate hydrogel comprises between 4% to 7% by weight of zinc ions and 3% to 6 % by weight of calcium ions. 2. - A scaffold according to claim 1, characterized in that the calcium and zinc alginate hydrogel integrated into the body, support or porous structure is arranged as a coating or filling of the structure or porous body of polymeric material. 3. - A scaffold according to any of the preceding claims, characterized in that the body, support or porous structure with a three-dimensional (3D) shape is made from a polymeric material selected from the group comprising: aliphatic polyesters, poly(amino acids ), copoly(ether esters), polyalkylene oxalates, polyamides, polycarbonates derived from tyrosine, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amino groups, poly(anhydrides), polyphosphazenes, biomolecules such as collagen, elastin, hyaluronic acid, bioabsorbable starches, and mixtures thereof, aliphatic polyesters, such as, homopolymers and copolymers of lactide, including lactic acid, D-, L- and mesolactide, glycolide, including glycolic acid), ε-caprolactone, p- dioxanone (1,4-dioxan-2-one), trimethylene carbonate (1,3-dioxan-2-one), alkyl derivatives of trimethylene carbonate, δ-valerolact one, ?-butyrolactone, ?-butyrolactone, ?-decalactone, hydroxybutyrate (repeating units), hydroxyvalerate (repeating units), 1,4-dioxepan-2-one, including its 1,5,8,12-dimer tetraoxacyclotetradecane-7,14-dione, 1,5-dioxepan-2-one, 6,6-dimethyl-1,4-dioxan-2-one, 2,5-diketomorpholine, pivalolactone, alpha, alpha-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-1,4-dioxan-2,5-dione, 3,3-diethyl-1,4-dioxan-2,5-dione, 6,8-dioxabicycloctan-7- one and polymer mixtures thereof. 4. - A scaffold according to claim 3, characterized in that the body, support or porous structure with a three-dimensional (3D) shape is made from polylactic acid (PLA). 5. - A scaffold according to any of the preceding claims, characterized in that the calcium and zinc alginate coating comprises between 1.5 - 2% w/w (with respect to the total weight of the scaffold) 6. - A scaffold according to any of the preceding claims characterized in that the pore volume is 20 to 30% with an average pore size of 400 to 600 pm. 7. - A scaffold according to any of the preceding claims, characterized in that it optionally incorporates components selected from the group comprising: graphene, graphene oxide, carbon nanofibers, graphite, reduced graphene oxide, reduced graphene, carbon nanotubes of single or multi-walled, fullerene, alkylamine- or ammonia-functionalized graphene oxide, boron-doped graphene, nitrogen-doped graphene, phosphorus-doped graphene, sulfur-doped graphene, boron-nitrogen doped graphene, phosphorus-nitrogen doped graphene , sulfur and nitrogen doped graphene and sulfonated reduced graphene oxide, nanocomposites with carbon nanomaterials such as graphene/TiO2, graphene/Fe3O4, graphene/Mn3O4, graphene/Pd, graphene/Pt, graphene/PtCo, graphene/PtPd, graphene reduced graphene/TiO2, reduced graphene/Fe3O4, reduced graphene/Mn3O4, reduced graphene/Pd, reduced graphene/Pt, reduced graphene/PtCo and reduced graphene/PtPd and poly nanoparticles pyrrole, nanoparticles and/or nanofibers such as silver or gold nanoparticles, nanocellulose, or ceramic nanoclays. 8. - A process for obtaining a scaffold according to claims 1 to 7 comprising the following stages: a) 3D printing of a support, structure or polymeric porous body (1); b) Vacuum immersion of the structure in an aqueous solution of sodium alginate (2,3); c) Sonication (4); d) Immersion of the sodium alginate polymer structure in an aqueous solution of calcium chloride (5,6); e) Immersion of the calcium alginate polymer structure in an aqueous solution of zinc chloride (7.8) f) Purification or washing of the scaffold (10); and g) Oven drying (9). 9. - A process according to claim 8, characterized in that the 3D printing of the support, structure or porous body is carried out with the fused deposition modeling technique. 10. - A process according to claims 8 - 9, characterized in that the vacuum immersion of the structure in stage b) is carried out in an aqueous solution of sodium alginate at a concentration of 2% w/v. 11. - A process according to claims 8 - 10, characterized in that the immersion of the structure in stage d) is carried out in an aqueous solution of calcium chloride at a concentration of 0.5% w/v and the immersion of the structure in step e) is carried out in an aqueous solution of zinc chloride at a concentration of 0.5% w/v. 12. - A scaffold according to any of claims 1 to 7 for use in reconstructive processes of bone defects such as: segmental defects, defects caused by trauma, osteolytic tumors, gunshot wounds, defects caused by osteoarthritis, surgery , osteomyelitis, tumor surgery, structural bone defects, defects caused by osteoporosis, osteopenia and/or metabolic bone disease. 13. - An implant, graft and bone graft substitute comprising a scaffold according to any of claims 1 to 7 and 12.