Large 3D porous scaffolds made of active hydroxyapatite obtained by biomorphic transformation of natural structures and process for obtaining them

Abstract

The present disclosure relates to a hydroxyapatite obtained from porous wood, having high compressive strength and dimensions suitable for clinical applications. The porous wood has a porosity of between about 60% and about 95%, said porosity being measured after subjecting the wood to a step of pyrolysis, and is selected from among rattan, pine, abachi, balsa, sipo, oak, rosewood, kempas and walnut wood. The hydroxyapatite may be substituted with one or more ions such as magnesium, strontium, silicon, titanium, carbonate, potassium, sodium, silver, gallium, copper, iron, zinc, manganese, europium, gadolinium. Also disclosed is a bone substitute comprising hydroxyapatite obtained from porous wood. The bone substitute is utilized for the substitution and regeneration of a bone or a bone portion, preferably for bones subjected to mechanical loads, such as long bones of the leg and arm, preferably the tibia, fibula, femur, humerus and radius. The invention relates also to a process for manufacturing a biomorphic hydroxyapatite scaffold from wood.

Claims

1. A method of treating of a human or animal bone having a bone defect, comprising the steps of: providing a bone substitute comprising a biomorphic hydroxyapatite scaffold obtained from a wood having a total porosity of at least 20%, said porosity being measured after subjecting the wood to a step of pyrolysis, said scaffold having a length, measured along a direction in which a dimension of the scaffold is maximum, greater than or equal to 2 cm, wherein the bone substitute has a shape that corresponds to the shape of a bone defect; implanting the bone substitute in the bone defect of the patient.

2. The method according to claim 1, further comprising the steps of providing a 3D model of the bone defect and, based on the 3D model obtained, imparting to the scaffold a shape corresponding to the shape of the bone defect.

3. The method of claim 1, wherein said human or animal bone is a bone subjected to mechanical loads.

4. The method of claim 1, wherein said human or animal bone is tibia, metatarsus, fibula, femur, humerus or radius.

5. The method of claim 1, wherein said human or animal bone is a cranial bone-part, a spine bone or a maxilla-facial bone-part.

6. The method of claim 1, wherein the total porosity of the wood is between 60% and 95%.

7. The method of claim 1, wherein the biomorphic hydroxyapatite scaffold has a compressive strength, measured in the longitudinal direction, greater than 5 MPa.

8. The method of claim 1, wherein the biomorphic hydroxyapatite scaffold has a hierarchically organized pore structure derived from the hierarchically organized pore structure of the wood from which it is obtained.

9. The method of claim 1, wherein the wood is selected from the group consisting of rattan, pine, abachi, balsa, sipo, oak, rosewood, kempas and walnut wood.

10. The method of claim 1, wherein the hierarchically organized pore structure includes between 30 and 80%, of the total porosity, of pores having a diameter below 150 μm, the reminder to 100% of total porosity being pores having diameter greater than 150 μm.

11. The method of claim 1, wherein the wood is rattan, and between 30% and 60% of the total porosity of the scaffold is due to pores having a diameter≤10 μm.

12. The method of claim 1, wherein the wood is rattan, and at least 25% of the total porosity of the total porosity of the scaffold is due to pores having a diameter≤1 μm.

13. The method of claim 1, wherein the wood is rattan, and at least 20% of the total porosity of the scaffold is due to pores that have diameter≥150 μm.

14. The method of claim 1, wherein the biomorphic hydroxyapatite scaffold has a specific surface area (SSA)>9 m2/g.

15. The method of claim 1, wherein the hydroxyapatite is partially substituted with one or more ions selected from the group consisting of magnesium, strontium, silicon, carbonate, sodium, potassium, silver, gallium and copper.

16. A method of treating of a human or animal bone having a bone defect, comprising the steps of: providing a bone substitute comprising a biomorphic hydroxyapatite scaffold obtained from a wood having a total porosity of between 60% and 95%, said porosity being measured after subjecting the wood to a step of pyrolysis, wherein said hydroxyapatite comprises a hierarchically organized pore structure and a compressive strength, measured along the longitudinal direction, greater than 5 MPa, wherein the bone substitute has a shape that corresponds to the shape of a bone defect; implanting the bone substitute in the bone defect of the patient.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) The following figures illustrate preferred embodiments of the subject matter disclosed herein. The claimed subject matter may be understood by reference to the following description taken in conjunction with the accompanying figures, in which:

(2) FIG. 1 shows a particular embodiment of the biomorphic scaffold that features a central channel.

(3) FIG. 2 shows photographs depicting tissues of explanted mice calvaria after 8 weeks. Extensive bone formation and penetration of the scaffold pores was achieved to a similar extent when implanted alone or with osteogenic stromal cells, where a) represents wood and no cells, b) represents hydroxyapatite and no cells, c) represents wood and cells, d) represents hydroxyapatite and cells, and where e) represents the scaffold implanted in a critical size defect of a mouse calvaria. The arrows in FIG. 2 f) point to the channel-like pores of the scaffold shown in FIG. 2 e) which mimic Haversian systems. Haversian systems or osteons are the functional unit of compact bone, in the form of cylinders made of bone lamellae organized in concentric layers. In the middle of the osteons a channel exist, containing the bone's nerve and the blood supply.

(4) FIG. 3 shows a stress-strain curve of a biomorphic scaffold subjected to compressive loading, wherein y=stress (N), and x=strain (mm).

(5) FIG. 4 shows the distribution of internal pore volume of the biomorphic hydroxyapatite of the present disclosure compared with the pore volume of the hierarchically structured hydroxyapatite obtained with a method known in the art.

(6) FIG. 5 shows a graph depicting the enhanced viability of mesenchymal stem cells when in contact with scaffolds comprising 2 and 5 mol % of Sr, in comparison with a strontium-free scaffold (BC) after 24 hours, 48 hours, 72 hours, 7 days and 14 days. y=% respect to BC.

(7) FIG. 6 shows graphs depicting the expression of osteogenesis-relevant genes, such as (a) RUNX2 and (b) ALP in scaffolds containing 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of strontium. y=fold-change expression relative to BC and x=days.

(8) FIG. 7 shows a graph depicting osteoblast viability when in contact with scaffolds comprising 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of Sr, in comparison with a strontium-free scaffold (BC) after 24 hours, 48 hours, 72 hours, 7 days and 14 days. y=% respect to BC.

(9) FIG. 8 shows graphs depicting the expression of osteogenesis-relevant genes, such as (a) Osterix, (b) BGlap, and c) IBSP in scaffolds containing 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of strontium. y=fold-change expression relative to CT and x=days.

(10) FIG. 9 shows graphs depicting the expression of osteoclast-relevant genes, such as (a) Osacr, (b) CTSK, and c) Itg β3 in scaffolds containing 2 mol % (Sr2%-BC) and 5 mol % (Sr5%-BC) of strontium. y=told-change expression relative to CT and x=days. A significant decrease of the genes involved in the principal molecular pathways of osteoclasts over time can be seen; thus indicating that the presence of Sr.sup.2+ ions in the scaffold inhibits osteoclast formation and activity; in figure a) the 14 day data were below the detection limit.

(11) FIG. 10 shows a comparison of the pore distribution of two calcium carbide scaffolds obtained after the carburization step of the prior art and the carburization step of the present invention, further compared with the pore distribution of the starting pyrolized wood. The specific surface area of the two calcium carbide scaffolds are also reported in the figure;

(12) FIGS. 11, 12 and 13—top two pictures show SEM images of two calcium carbide scaffolds obtained with the process of the invention and the prior art process, respectively;

(13) FIG. 13—bottom two pictures show a comparison of the dimension of the carcium carbide granes;

(14) FIG. 14 show a comparison of the crystal phase of the two calcium carbide scaffolds, measured with x ray-XRD, obtained with the process of the invention and the prior art process, respectively.

(15) FIG. 15 shows SEM images of the calcium oxide scaffold obtained after the oxidation step of the present invention and the prior art oxidation step;

(16) FIG. 16 depicts the pore distribution of the two calcium oxide scaffolds obtained after the oxidation step of the present invention and the prior art oxidation step, respectively;

(17) FIGS. 17 and 18 show SEM images of the calcium carbonate obtained after the carbonation step according to the present invention and the prior art carbonation step, respectively;

(18) FIG. 19 shows that the pore distribution of the two calcium carbonate scaffolds obtained after the carbonation step according to the present invention and the prior art carbonation step, respectively;

(19) FIG. 20 shows the comparison between the pore distribution of the final biomorphic hydroxyapatite scaffold (after phosphatization) obtained with the process of the disclosure and the final biomorphic hydroxyapatite scaffold of the prior art;

(20) FIG. 21 shows the result obtained after subjecting a piece of rattan wood having a length, measured along a direction in which a dimension of the scaffold is maximum, equal or greater than 2 cm, to the process steps according to the conditions described in the prior art: even before the phosphatization step the scaffold can break down;

(21) FIG. 22 shows the result obtained alter subjecting a piece of rattan wood having a length, measured along a direction in which a dimension of the scaffold is maximum, equal or greater than 2 cm, to the process steps according to the conditions described in the prior art: even if the scaffold survives the process steps up to phosphatization, after phosphatization the scaffold breaks down;

(22) FIG. 23 shows the relative quantification (2.sup.−ΔΔCt) of gene expression with respect to the expression of the not-doped prior art scaffold used as calibrator, after 14 days of mMSCs 3D cultured in dynamic condition with all the tested samples;

(23) FIG. 24 shows an embodiment of the biomorphic scaffold with cuboid shape.

(24) FIG. 25 shows that the channel-like structure of the biomorphic hydroxyapatite scaffold obtained with the process of the present disclosure is uniquely characterized by pervious large channels (100-300 micron in diameter) (micro CT Scan). Such channels are permissive to the formation of suitable blood vessels supporting bone regeneration.

DETAILED DESCRIPTION OF THE DISCLOSURE

(25) As used in present description and in the appended claims, “hierarchical pore structure” or “hierarchically organized pore structure” indicates a material having an anisotropic three dimensional pore structure in which channel-like pores are interconnected through transversal channels and wherein micro and nano-pores are present in the area surrounding the channel-like pores.

(26) As used in the present description and the appended claims, the values of “compressive strength” are obtained with the method described in the following description by exerting mechanical forces along the longitudinal and transversal directions, with respect to the channel-like pores.

(27) As used in the present description and the appended claims, “bone defect” refers to a missing part or portion of the bone or to the entire bone that is missing and needs to be totally replaced by the scaffold of the disclosure.

(28) As used in the present description, “biomorphic hydroxyapatite” refers to a material that: 1) consists of hydroxyapatite or 2) comprises hydroxyapatite or 3) is a material comprising or consisting of hydroxyapatite and tricalcium phosphate. In case the material consists of hydroxyapatite and tricalcium phosphate, the material is a biphasic material. In an embodiment of the invention, when the “biomorphic hydroxyapatite” is a material comprising or consisting of hydroxyapatite and tricalcium phosphate, the hydroxyapatite is doped with one or more ions chosen in the group consisting of magnesium, strontium, silicon, titanium, carbonate, potassium, sodium, silver, gallium, copper, iron, zinc, manganese, europium, gadolinium and mixtures thereof.

(29) The inventors of the present patent application have surprisingly found that it is possible to obtain a biomorphic hydroxyapatite scaffold from wood which exhibits a biomimetic chemical composition, an adequate mechanical performance, a morphology that is favorable to cell colonization and vascular growth and, at the same time, has dimensions that are suitable for clinical applications.

(30) In a first aspect, the present disclosure describes a biomorphic hydroxyapatite scaffold obtained from a wood having a total porosity of at least 20%, preferably at least 40%, more preferably comprised between 60% and 95%, wherein said porosity is measured after subjecting the wood to the step of pyrolysis, the scaffold having a length, measured along a direction in which a dimension of the scaffold is maximum, greater than or equal to 2 cm.

(31) The total porosity of the biomorphic hydroxyapatite scaffold obtained after the process of the disclosure is the same as the total porosity of the starting wood measured after subjecting the wood to the step of pyrolysis. In particular, the total porosity of the biomorphic hydroxyapatite scaffold obtained after the process of the disclosure is at least 20%, preferably at least 40%, more preferably comprised between 60% and 95%.

(32) Preferably, the scaffold has a length, measured along a direction in which a dimension of the scaffold is maximum, that is greater than or equal to 2 cm and reaches an end value that is determined according to the clinical application. For example in the case of bone substitution of long bones, such as tibia, femur, fibula, humerus, radius, the length of the scaffold, measured along a direction in which a dimension of the scaffold is maximum, can be comprised between 2 and 20 cm.

(33) Preferably, the scaffold of the disclosure has a compressive strength measured in the longitudinal direction of greater than 5 MPa, preferably between 10 MPa and 20 MPa.

(34) Preferably the scaffold of the disclosure shows a compressive strength along the transversal direction of up to 10 MPa.

(35) Preferably, the biomorphic hydroxyapatite scaffold is characterized by a hierarchically organized pore structure.

(36) The “hierarchical pore structure” or “hierarchically organized pore structure” of the hydroxyapatite scaffold of the disclosure derives from the complex three-dimensional hierarchical structure of the starting wood from which the scaffold is obtained and therefore has a range of differently sized pores. The differently sized pores in the hierarchical structure render it desirable for use as a bone substitute.

(37) For example, pores having diameter≥200 μm, preferably between 150-300 μm, more preferably 200-300 μm will permit cell colonization and proliferation and the formation of an appropriate vascularization tree. Pores having a diameter s 10 μm, preferably <1 μm, more preferably between 0.01 and 0.1 μm (micro and nano-pores), that partially interconnect the channel-like pores, permit exchange of nutrient fluids and discharge of the waste products of cell metabolism.

(38) The preservation of the hierarchical pore structure of wood in the hydroxyapatite, provides scaffolds with optimal mechanical features and enables the efficient discharge of mechanical loads.

(39) As hydroxyapatite obtained from wood reproduces the structure of a natural material in detail, it can be thus referred to as being biomorphic.

(40) In particular, the hierarchically organized pore structure of the scaffold of the disclosure includes between 30% and 80% (of the total porosity) of pores having a diameter below 150 μm, the reminder to 100% of total porosity being pores having diameter greater than 150 μm.

(41) In one embodiment, preferably when the starting wood is rattan, between 30% and 60% of the total porosity of the scaffold is due to pores having a diameter≤10 μm.

(42) In one embodiment, preferably when the starting wood is rattan, at least 25% of the total porosity, preferably between 25% and 50% of the total porosity of the hydroxyapatite scaffold is due to pores having a diameter≤1 μm, preferably ≤0.1 μm, in particular between 0.01 and 0.1 μm.

(43) In one embodiment, preferably when the starting wood is rattan, at least 20% of the total porosity of the hydroxyapatite scaffold is due to pores that have diameter≥150 μm.

(44) Preferably, the hydroxyapatite scaffold has a specific surface area (SSA)>9 m.sup.2/g, preferably from 9 to 20 m.sup.2/g.

(45) The wood used to obtain the hydroxyapatite scaffold can be any wood having a total porosity of at least 20%, preferably at least 40%, more preferably comprised between 60% and 95%, even more preferably a porosity of between 65% and 85% (said porosity being measured after subjecting the wood to a step of pyrolysis).

(46) Examples of suitable woods used to obtain the hydroxyapatite include rattan, pine, abachi, balsa, sipo, oak, rosewood, kempas and walnut wood, preferably the wood used is rattan wood.

(47) The hydroxyapatite scaffold obtained from wood may comprise hydroxyapatite which is partially substituted with one or more ions. Examples of such ions are carbonate, magnesium, strontium, silicon, titanium, sodium, potassium, silver, gallium, copper, iron, zinc, manganese, europium and gadolinium. The introduction of carbonate in the phosphate site increases bio-solubility and enhances surface affinity to osteoblast cells.

(48) The introduction of magnesium provides enhanced ability of new bone apposition and formation. The introduction of strontium assists in re-establishing bone production, affected by metabolic diseases such as osteoporosis, so that its presence may enhance bone regeneration.

(49) The introduction of silver, gallium, copper and zinc provides antibacterial properties. When the hydroxyapatite scaffold obtained from wood comprises hydroxyapatite which is partially substituted with one or more ions, the scaffold is a material comprising or consisting of doped hydroxyapatite and tricalcium phosphate. According to a further embodiment, the biomorphic hydroxyapatite scaffold of the instant disclosure may comprise: 0-15 wt % of magnesium, preferably 1-10 wt %; and/or 0-15 wt % of carbonate, preferably 1-10 wt %; and/or 0-15 wt % of strontium, preferably 1-10 wt %; and/or 0-20 wt % of titanium, preferably 1-10 wt %; and/or 0-15 wt % of potassium, preferably 1-10 wt %; and/or 0-15 wt % of sodium, preferably 1-10 wt %; and/or 0-15 wt % of silicon, preferably 1-10 wt % and/or; 0-15 wt % of silver, preferably 1-10 wt % and/or; 0-15 wt % of gallium, preferably 1-10 wt % and/or; 0-15 wt % of copper, preferably 1-10 wt % and/or; 0-30 wt % of iron, preferably 1-10 wt %; and/or 0-15 wt % of zinc, preferably 1-10 wt % and/or; 0-15 wt % of manganese, preferably 1-10 wt % and/or; 0-15 wt % of europium, preferably 1-10 wt % and/or; 0-15 wt % of gadolinium, preferably 1-10 wt % and/or.

(50) The biomorphic hydroxyapatite scaffold obtained from wood according to the present disclosure, has bioactivity and bioresorbability characteristics combined with mechanical strength characteristics and dimensions that makes it particularly suited for clinical use as a bone substitute, in particular in humans and animals. Such a bone substitute could be used to substitute and/or reconstruct and/or regenerate bone, bone portions or bone defects. For example, the bone substitute could be used to substitute or regenerate bone or bone portions that are subjected to mechanical loads. For example, the bone substitute could be used to substitute or regenerate long bones of the arms and legs. Such long bones could include the tibia, femur, fibula, humerus, radius, etc.

(51) The bone substitute could also be used in the substitution and/or reconstruction of cranial bone-parts, maxillofacial bone-parts and spine bones e.g. vertebral bodies, intervertebral disc) and in spinal fusion surgery procedures.

(52) When used as a bone substitute, the biomorphic hydroxyapatite scaffold can have a shape that adapts to the shape of the bone defect that needs to be reconstructed in such a way as to substantially fill the bone gap. Therefore, the scaffold and the bone substitute of the disclosure can have any shape that is suitable for the purpose of reconstructing and regenerating a bone defects or for substituting any missing part of the bone.

(53) For example the scaffold or the bone substitute of the present disclosure may take the form of a cylinder, right prism, or cuboid, or wedges. In one embodiment, the scaffold or the bone substitute comprises a central channel with a diameter of between about 20% to about 60% of the diameter of the scaffold or the bone substitute. In particular, the scaffold or the bone substitute has a tubular shape.

(54) In one embodiment, the present disclosure further refers to a scaffold or a bone substitute having a cylindrical, right prism, cuboid or tubular shape, having a height greater than or equal to 2 cm.

(55) In an embodiment of the present disclosure, the scaffold or the bone substitute may be coated with a thin layer based on hydroxyapatite and/or collagen to increase cellular adhesion and proliferation, and thus osteointegration in the surrounding bone tissue. The layer may additionally comprise hydroxyapatite substituted with one or more ions relevant for the stimulation of the bone regeneration such as carbonate, magnesium, silicon, potassium, sodium and strontium, or with antibacterial effect such as gallium, silver, copper or zinc.

(56) In a further embodiment of the present disclosure, the scaffold or the bone substitute may be soaked in a natural polymer (chosen among the group comprising gelatin, collagen, alginate, chitosan, gellan, cellulose) to further increase mechanical properties and further promoting cell adhesion.

(57) To the scaffold or bone substitute cells, platelet rich plasma, antibodies, growth factors proteins, DNA fragments, miRNA, siRNa can be added in order to help cell adhesion.

(58) Also drugs, such as antibiotics or anticancer drugs, can be added to the scaffold or bone substitute.

(59) The disclosure refers also to a method of reconstruction and/or regeneration of a human or animal bone having a bone defect, comprising the steps of: providing a bone substitute comprising or consisting of the biomorphic hydroxyapatite scaffold of the disclosure having a shape that corresponds to the shape of a bone defect; implanting the bone substitute in the bone defect of the patient.

(60) Preferably, the method of reconstruction and/or regeneration includes the steps of providing a 3D model of the bone defect and, based on the 3D model obtained, imparting to the scaffold a shape corresponding to the shape of the bone defect. The step of imparting a shape to the scaffold can be applied to the starting piece of wood or to the hydroxyapatite scaffold obtained at the end of the transformation process of the disclosure or to the scaffold obtained after each step of the process (e.g. after the carbonation step). Preferably the step of imparting the shape is applied on the starting piece of wood.

(61) The biomorphic hydroxyapatite scaffold of the disclosure and the biomorphic hydroxyapatite scaffold partially substituted with one or more ions, is obtained from a multistep transformation process comprising the following steps:

(62) 1) Pyrolysis: a native wood is heated at a temperature in the range of 600° C. to 1000° C. under an inert atmosphere to permit the decomposition and the elimination of all organic substances. From this process, a carbon template is obtained.

(63) 2) Carburization: the carbon template is infiltrated with calcium in the vapour state at a temperature in the range 900 to 1200° C., and at a pressure<1000 mbar, preferably <600 mbar, more preferably in the range of 0.05 to 100 mbar, thus transforming the carbon template into calcium carbide (CaC.sub.2).

(64) 3) Oxidation: the calcium carbide template is heated in air at a temperature in the range of 750 to 1300° C., preferably 1000-1200° C., thus enabling the transformation of calcium carbide into calcium oxide (CaO).

(65) 4) Hydration: the calcium oxide template is exposed to water, thus enabling water uptake in an amount of 1-25 mole %, preferably 5-15 wt %.

(66) 5) Carbonation: the calcium oxide template is transformed into calcium carbonate by heating at a temperature in the range of 500 to 900° C., preferably at a temperature in the range of 750 to 850° C. under a CO.sub.2 pressure, or a mixture of CO.sub.2 and an inert gas (e.g. argon, nitrogen) pressure. The pressure range is from 4 to 20 MPa.

(67) 6) Phosphatization: the calcium carbonate template is treated with at least one phosphate salt.

(68) In the pyrolysis step 1) of the multi-step process, the native wood is preferably selected among rattan, pine, abachi, balsa sipo, oak, rosewood, kempas and walnut wood. More preferably the native wood is rattan wood.

(69) The native wood has a total porosity of at least 20%, preferably at least 40%, more preferably comprised between 60% and 95%, wherein said porosity is measured alter subjecting the wood to the step of pyrolysis.

(70) Prior to the pyrolysis step 1), the starting native wood may be optionally dried at a temperature of between 50° C. and 90° C., preferably at a temperature of between 60° C. and 80° C., more preferably at a temperature of between 65° C. and 75° C. The native wood may be dried for more than 6 hours, preferably for more than 12 hours, preferably for more than 18 hours, preferably for a time comprised between 20 and 30 hours.

(71) In the pyrolysis step of the multi-step process, the inert atmosphere may be an atmosphere of a gas selected from the group comprising nitrogen and argon.

(72) In the pyrolysis step of the multi-step process, the native wood may be heated at a temperature of between 600° C. to 1000° C., preferably at a temperature of between 800° C. and 1000° C. The pyrolysis step may last more than 6 hours, preferably more than 12 hours, preferably more than 18 hours, preferably said step may last for a time comprised between 20 and 30 hours.

(73) The thermal cycle of the pyrolysis step 1) may be carried out by heating the native wood at the rate not higher than 5° C./min, preferably not higher than 3° C./min and by cooling at a rate not higher than 3° C./min, preferably not higher than 2° C./min, to prevent crack formation and internal fracture of the material.

(74) Prior to the pyrolysis step 1), the multistep method can additionally comprise a step i) of selection and preparation of the native wood, wherein said native wood can be cut into a piece having a shape corresponding to the shape of a bone defect to be reconstructed. In particular the native wood is shaped into a piece of wood having a length, measured along a direction in which a dimension of the wood is maximum, that is greater than or equal to 2 cm. Preferably, the dimension of the wood reaches an end value that is determined according to the clinical application.

(75) For example, the native wood can be shaped in the form of a cylinder, right prism, or cuboid. The native wood can also be shaped in such a way as to comprise a central channel with a diameter of between about 20% to about 60% of the diameter of the piece of wood. In particular, the native wood can be cut into a tubular shape.

(76) Preferably, step i) of selection and preparation of the native wood, comprises the steps of: providing a 3D model of a bone defect and, based on the 3D model obtained, imparting to the native wood a shape corresponding to the shape of the bone defect. The step of imparting a shape to the native wood can be applied to the starting native wood or to the hydroxyapatite scaffold obtained at the end of the multi-step transformation process. Preferably the step of imparting the shape is applied on the starting native wood to avoid internal and external damage (fracture) of the scaffold.

(77) In the carburization step 2) of the multi-step process, the reaction is preferably carried out with a Ca/C molar ratio (at the beginning of the reaction) in the range of 1.10 to 2.50, preferably in the range of 1.50 to 2.00. The Ca/C molar ratio is important because ratios below the range lead to incomplete reactions and ratios above the range lead to obstruction of the pores by residues of Ca.

(78) In the carburization step of the multi-step process, the carbon template is heated at a heating rate in the range of 1 to 10° C./min, preferably at a heating rate in the range of 1 to 7° C./min.

(79) The inventors of the present patent application have surprisingly found that carrying out the carburization step at a reduced pressure as above described is an advantage for the successful application of the subsequent process steps, particularly when large biomorphic scaffold needs to be produced.

(80) In fact, by using the above-described pressure conditions, the evaporation of calcium can occur at temperatures that are about 400-500° C. lower than the boiling point of calcium at room atmosphere (i.e. 1484° C.), thus yielding, unexpectedly, the complete transformation of the pyrolized wood into calcium carbide at a temperature much lower than any other process known in the art. In particular, the use of pressure in the range of 0.5-600 mbar, or preferably 0.05 to 100 mbar, results in a substantially complete transformation of the pyrolized wood into calcium carbide.

(81) A substantially complete transformation of the pyrolized wood into calcium carbide will result in an advantage for the yields of the subsequent transformation steps.

(82) The carburization conditions of the present disclosure also improve preservation of the micro- and nano-pores having diameters≤1 μm (preferably from 0.01 to 0.1 μm) of the native wood both in the scaffold after carburization and in the final biomorphic scaffold, with respect to the known scaffolds obtained from wood using known processes.

(83) The carburization phase is a critical step in the process because a good preservation of the micro- and nano-porosity after this step will ensure that the final biomorphic scaffold exhibits similar nano/micro-porosity. The presence of a high percentage of well interconnected micro and nano-pores in the final biomorphic scaffold permits exchange of nutrient fluids and discharge of the waste products of cell metabolism.

(84) Besides improving the preservation of the micro- and nano-pores having diameters≤1 μm, the carbonization conditions here described yield a scaffold after carburization (and also a final biomorphic scaffold) with specific surface area (SSA) from 9 to 20 m.sup.2/g. Such a specific surface area is about 2-fold higher than the SSA of a scaffold obtained with a process known in the art—which is about 5-6 m.sup.2/g (see comparative example 4 and FIGS. 10-13).

(85) The carburization conditions of the present disclosure also yield a porous calcium carbide scaffold containing calcium carbide grains that are smaller than the grains of a scaffold obtained with a known process (see comparative example 4 and FIG. 13—bottom pictures).

(86) The comparative examples show that the dimensions of the calcium carbide grains in the scaffold after carburization according to the present disclosure is about 5-15 μm (preferably about 10 μm), while the crystal dimensions of the scaffold after carburization obtained with known processes is about 100 μm.

(87) The inventors of the present patent application have surprisingly found that the higher specific surface area (SSA) and the smaller dimension of the grains that are obtained with the carburization conditions above-described, and could not obtained by previously disclosed methods, is important to ensure high yield of transformation of the native wood after each step of the process.

(88) Comparative example 4 and FIG. 14 also show that the scaffold after carburization according to the present disclosure contains calcium carbide with a mixture of tetragonal and cubic crystal lattice, while the scaffold obtained with known processes contains calcium carbide with a tetragonal crystal lattice only. Since calcium oxide has a cubic crystal structure only, the transformation from a calcium carbide, which is partially in a cubic form, to calcium oxide, can occur with a lower risk of generating microfractures in the scaffold. This is extremely advantageous for the quality of the final biomorphic scaffold.

(89) Therefore, the conditions employed in the carburization step strongly reduce the number of defects that can be observed in the hierarchical pore structure of the calcium carbide.

(90) In addition, the low temperatures employed in the present disclosure (i.e. well below 1500° C.) prevent grain coalescence and excessive consolidation of the calcium carbide, which provoke structural distortion and deviations from the original microstructure of the starting wood, thus impairing the outcome of the following process steps.

(91) In the oxidation step 3) of the multi-step process, the calcium carbide template may be heated to a final temperature in the range of 800 to 1300° C., preferably to a final temperature in the range of 1000 to 1200° C.

(92) In the oxidation step, the calcium carbide template may be heated at a heating rate in the range of 1 to 15=C/min, preferably at a heating rate in the range of 1 to 7° C./min. The oxidation of calcium carbide obtained under pressure according to the carburization step described above leads to a scaffold of calcium oxide with higher specific surface area (SSA) and a porosity with a higher micro- and nano-pores fraction with respect to scaffolds obtainable with the known processes (see comparative example 4 and FIG. 16). The comparative experiment show that the micro and nano-porosity is conserved also after the oxidation step.

(93) In the hydration step 4) of the multi-step process the calcium oxide template is exposed to water, thus enabling water uptake in an amount preferably comprised in the range of 1-25 mole %, more preferably comprised in the range 5-15 mole %. This step leads to the formation of hydrated calcium oxide containing calcium hydroxide in amount≤50% by weight of the 3D structure, that catalyzes the subsequent carbonation of CaO. The amount of calcium hydroxide (Ca(OH).sub.2) as intermediate product must be strictly controlled to avoid the collapse of the 3D structure. The hydration conditions here described allow to keep the amount of calcium hydroxide s 50%.

(94) In a preferred embodiment, the hydration step is conducted at the same time as the carbonation step, for example by using CO.sub.2 enriched with water.

(95) In the carbonation step 5) of the multi-step process, the use of high temperature while progressively increasing the CO.sub.2 pressure in the system up to the values indicated above, surprisingly enables substantially complete conversion of hydrated calcium oxide into a calcium carbonate template which exhibits surprisingly high cohesion and mechanical strength.

(96) The carbonation step of the multi-step process may be carried out according to one of the following thermal cycles: at a constant CO.sub.2 pressure of about 10-15 MPa, slowly increasing the temperature at a value in the range of about 750-850° C., preferably at about 800° C.; at a constant temperature of about 750-850° C. (or about 700-800° C.), preferably at about 800° C. raising the pressure up to about 10-15 MPa; keeping the pressure at about 4-6 MPa while raising the temperature up to about 750-850° C. (or about 700-800° C.), preferably up to about 800° C. and subsequently increasing the pressure up to about 10-15 MPa.

(97) The carbonation process occurs through the formation of reactive intermediates, such as calcium hydroxide. This leads to a final calcium carbonate characterized by a fine-grained structure substantially free of any large cubic crystal of calcium carbonate (>10 μm), which might compromise the structural integrity of the 3D structure. Comparative example 4 and FIGS. 17-18 show that, thanks to the application of a hydration step, after the carbonation step a finer-grained structure is obtained, compared to the intermediate 3D structure that is obtained with the processes known in the art.

(98) The carbonation step carried out in the conditions described above results in superior mechanical properties of the biomorphic hydroxyapatite scaffold obtainable by the process of the disclosure when compared to similar processes known in the art, in which the carbonation step is carried out at high temperature and low pressure or at high pressure and low temperature.

(99) The inventors of the present patent application have surprisingly found that the achievement of the above reported features in the calcium carbonate is an important condition to enable the complete transformation of large pieces (i.e. ≥2 cm) into a final biomorphic scaffold having the desired composition and maintenance of the original wood microstructure.

(100) In the phosphatization step 6) of the multi-step process, the at least one phosphate salt may be selected from the group consisting of ammonium phosphate, sodium phosphate, and potassium phosphate. The use of ammonium phosphate enables a better control of the pH, hence the conversion process is more efficient and the resulting body has favorable mechanical properties and physical cohesion.

(101) In the phosphatization step of the multi-step process, the calcium carbonate template may be immersed in a solution comprising at least one of said phosphate salts. The solution may have a concentration of 0.1 to 5M, preferably a concentration of 0.5 to 2.0M.

(102) The starting ratio of PO.sub.4/CO.sub.3 in the phosphatization step of the multi-step process is preferably 1.5 to 5 times the theoretical stoichiometric value, preferably 2 to 4 times the theoretical stoichiometric value.

(103) In the phosphatization step of the multi-step process, the calcium carbonate template immersed in a phosphate-rich solution may be heated from 25° C. to 300° C. under a vapour pressure in the range of 0.1 to 2.5 MPa (hydrothermal conditions).

(104) The phosphatization step may last about 12 to about 180 hours, preferably about 48 to about 120 hours, more preferably from 24 to 72 hours.

(105) The starting pH of the phosphate-rich solution in the phosphatization step of the multi-step method is preferably between pH 7 and 12.

(106) Substitution of the hydroxyapatite with other ions can be achieved by introducing suitable soluble salts containing the ions of interest during or after the process completion, preferably during the phosphatization process. Suitable ions may include strontium, magnesium, silicon, titanium, carbonate, sodium, potassium, gallium, silver, copper, iron, zinc, manganese, europium, gadolinium, and mixtures thereof. An example of a solution containing magnesium ions is MgCl.sub.2.6H.sub.2O, and an example of a solution containing strontium ions is SrCl.sub.2.6H.sub.2O.

(107) As a consequence of the ionic doping, the final biomorphic scaffold comprises or consists of a material comprising or consisting of doped hydroxyapatite and tricalcium phosphate.

(108) According to a less preferred embodiment, where the native wood has not been shaped into form and dimensions suitable for being used as bone substitute (i.e. if step i) is not carried out), the biomorphic hydroxyapatite scaffold obtained from the multi-step process may conveniently be shaped into a scaffold having the required form and shape by known techniques.

(109) The disclosure relates also to the biomorphic hydroxyapatite scaffold obtained (or obtainable) from the process described above, having improved physical and mechanical properties if compared to hierarchically structured hydroxyapatites obtained by similar processes known in the art.

(110) In particular, the biomorphic hydroxyapatite scaffold obtained (or obtainable) from the process of the present disclosure possess all the features above described for the scaffold or bone substitute of the disclosure.

(111) In particular, with respect to the prior art, the final biomorphic scaffold obtained with the process here described possess a porosity which is composed by a higher percentage of micro and nano-pores than the scaffolds obtained by similar processes known in the art. In particular at least 25% of the total porosity, preferably between 25% and 50% of the total porosity of the hydroxyapatite scaffold of the disclosure is due to pores having a diameter≤1 μm, preferably ≤0.1 μm, in particular between 0.01 and 0.1 μm.

(112) This high percentage of micro and nano-porosity is extremely advantageous from a clinical point of view because micro and nano-pores permit exchange of nutrient fluids and discharge of the waste products of cell metabolism enhance, thus improving bone regeneration.

(113) Moreover, the biomorphic scaffold of the disclosure shows a higher specific surface area (9 to 20 m.sup.2/g vs 5-6 m.sup.2/g) than a scaffold obtained with a process known in the art. A higher surface area determines enhanced surface bioactivity and enhanced wettability of the scaffold or bone substitute, thus improving the osteointegration and bio-resorption process.

(114) Also the biomorphic scaffold of the disclosure includes hydroxyapatite grains of about 100-200 nm (i.e. nano-grains), much smaller than the ones present in sintered hydroxyapatite (i.e. typically >1 pun). Small grains show a clinical advantage for bone regeneration because they can be more easily resorbed by the cells, thus allowing a better bone regeneration with respect to the scaffold known in the art.

(115) In addition, the biomorphic scaffold of the disclosure exhibits compressive strength greater than 5 MPa, preferably between 10 MPa and 20 MPa, measured in the direction along the channel-like pores (longitudinal direction), and a compressive strength along the direction of the transversal channels (transversal direction) of up to 10 MPa. The inventors of the present disclosure surprisingly found that these mechanical features make the final hydroxyapatite scaffold as a stand-alone material, therefore it can be applied in procedures of regeneration of load-bearing bone parts without the use of any reinforcing or sustaining structure such as shells or bars.

(116) In nature, nanocrystalline, ion-substituted hydroxyapatite is the main component present in hard body tissues; in fact, the mineral phase in bone is a nanostructured phase composed of finely dispersed hydroxyapatite platelets of dimensions below 100 nm that organize in a 3D hierarchically organized porous structure representing the whole bone tissue.

(117) In this respect, the inventors of the present disclosure surprisingly found that, when compared with previously known art, the biomorphic transformation of natural wood structures obtained by the above described process, can uniquely give rise to final hydroxyapatite bone scaffolds exhibiting simultaneously bone-mimicking composition, high open and interconnected macro/micro/nano-porosity and superior mechanical strength, associated with a size relevant for application in load-bearing sites, particularly in long segmental bones of the limbs, or large maxillofacial regions, or in spine.

(118) All these features, which have never been shown to occur simultaneously, are of outmost importance to enable extensive bone regeneration in load-bearing sites. The differences in the biomorphic hydroxyapatite scaffold structure obtainable with the process according to the present disclosure yield important clinical advantages that are shown in comparative example 6. In particular, the present scaffolds show a higher inductive power on the expression of osteogenic related genes, with respect to the prior art scaffolds, which translate in a better clinical performance in terms of bone regeneration.

(119) The present disclosure is further illustrated by the following, non-limitative, examples.

EXAMPLES

(120) Methods of Measurement

(121) Total Porosity of Wood Subjected to a Pyrolysis Step (Pyrolized Wood):

(122) A piece of pyrolyzed wood, shaped as a prism or a cylinder, is weighed, then the volume is obtained by measuring diameter and height. The absolute density (A.D.) of the pyrolyzed wood is obtained by weight/volume ratio; the relative density (R.D.) is obtained by dividing the A.D. of the pyrolyzed wood by the theoretical density of carbon (i.e. R.D.=A.D./2.25). The total porosity (%) is obtained by (1−R.D)*100.

(123) Total Porosity of a Scaffold Obtained after Each Step of the Process and of the Biomorphic Hydroxyapatite Scaffold Obtained at the End of the Process:

(124) The porosity is calculated by applying the same method as above using appropriate values for the theoretical density of each material obtained after each step (i.e. the theoretical density of CaC.sub.2, CaO, CaCO.sub.3, HA).

(125) Compressive Strength:

(126) the final scaffold or bone substitute, shaped as a prism or a cylinder, is subjected to loading by using a universal screw-type testing machine to obtain stress-strain curves and the fracture load. The compressive strength is given by the ratio between the fracture load and the area subjected to compression.

(127) Pore Diameter:

(128) the pore size distribution and pore morphology of the final scaffold or after each step of the process are evaluated by means of mercury intrusion porosimetry and scanning electron microscopy (SEM), respectively. Mercury intrusion porosimetry analysis is based on the measure of the intrusion of mercury into the pores of the sample at various pressures.

(129) Crystalline Phases: Identification and Quantification:

(130) of the crystalline phases on scaffolds are performed by X-ray powder diffraction technique (XRD), evaluating the result of the X-radiation incidence on the sample, with different and continuous angles.

(131) Specific Surface Area:

(132) the total surface area of the materials per unit of bulk volume (m.sup.2/g) is evaluated using the BET method, estimated from the amount of gas adsorbed in relationship with its pressure.

Example 1

Preparation of a Hydroxyapatite Derived from Wood Using the Multi-Step Process

(133) i) A piece of native rattan wood is shaped in a cylindrical form having the following dimensions: diameter=2 cm; height=3 cm;

(134) 1) Pyrolysis of the Native Wood

(135) The starting wood piece is dried at 70° C. for 24 hours, then treated at 800° C. for more than 30 minutes under flowing nitrogen, thus transforming into pure carbon template. Thermal cycle: heating at 1° C./min up to 350° C. and 2° C./min from 350° to 800° C. The sample is maintained at temperature of 800° C. for about at least 30 min and subsequently the template is cooled at 1° C./min.

(136) 2) Carburization

(137) The carbon template is subjected to heating at 1000° C. under argon and calcium atmosphere at 0.5 mbar, thus transforming in calcium carbide. Dwell time at 1000° C.=30 minutes.

(138) 3) Oxidation

(139) The calcium carbide template is heated in air up to 1100° C. following a heating rate in the range of 1−7° C./min, thus enabling the complete transformation into calcium oxide.

(140) 4) Hydration

(141) The calcium oxide body is activated by exposure to water, thus enabling water uptake in the amounts of about 10 mole %.

(142) 5) Carbonation

(143) The pre-conditioned hydrated body is heated to 800° C. under a progressively increasing CO.sub.2 pressure of 0.5 to 10 MPa. This transforms the calcium oxide body into calcium carbonate.

(144) 6) Phosphatization

(145) The calcium carbonate body is immersed in a 0.5 M ammonium phosphate solution and a starting PO.sub.4 to CO.sub.3 ratio of 2 times the theoretical stoichiometric value, at a temperatures of 200° C. under a water vapor pressure of 2 MPa.

(146) The compressive strength of the scaffold was evaluated by exerting mechanical forces along the perpendicular and transversal direction, with respect to the orientation of the channel-like pores.

(147) By loading along the pore direction (which is the most clinically-reflective configuration to mimic the in vivo biomechanical stimuli in the case of long segmental bones), the scaffold (developed as a hollow cylinder with outer diameter=15 mm; inner diameter=6 mm height=20 mm, and a pore extent of 60-65 vol %) exhibited compressive strength of up to 16 MPa (i.e. 250 Kg of ultimate load (FIGS. 1 and 3). In the transversal direction, the scaffold exhibited compressive strength of up to 4 MPa.

(148) The scaffold could also be subjected to thermal treatment at a maximum temperature of 1300° C., in a controlled atmosphere, to further increase the mechanical strength of the scaffold.

(149) The bone-like microstructural features of the biomorphic scaffold enables delivery of topological information to cells to build new bone tissue with organized structure. This was confirmed by in vivo tests where the scaffold was implanted in rabbit femurs and mouse calvaria.

(150) The scaffold did not induce any toxic adverse reactions nor any necrosis or infections after surgery. The scaffold yielded extensive colonization by the newly formed bone after 1 month, similar to the control which was a commercial porous apatite scaffold: EngiPore, Finceramica S.p.A., Italy.

(151) The tissues explanted from mice calvaria showed extensive bone formation and penetration into the scaffold pores both when the scaffold was implanted alone and also when osteogenic stromal cells were added to the implanted scaffold (FIG. 2a-d). The channel-like porosity of the scaffold induced the formation of bone structures mimicking Haversian systems (as indicated by the arrows in FIG. 2f). Moreover, the channel-like pores of the scaffold induced fast angiogenesis so to assist the formation and penetration of the new bone. This result confirms that a suitable orientation of the porosity in relationship to the orientation of the endogenous vascular network can be effective in promoting early development of extensive angiogenesis.

Example 2

Comparison of Biomorphic Hydroxyapatite of the Disclosure and Hierarchically Structured Hydroxyapatite Known in the Art

(152) A comparison test was made between the pore size distribution of biomorphic hydroxyapatite of the present disclosure obtained from rattan native wood and the pore size distribution of a hierarchically structured hydroxyapatite obtained from the same native wood according to the teaching of Anna Tampieri et al. in the Journal of Material Chemistry, 2009, 19, 4973-4980 using the teaching of Ruffini et al. in Chemical Engineering Journal 217 (2013) 150-158 for the phosphatization step only (Mixture of NH.sub.4H.sub.2PO.sub.4—(NH.sub.4).sub.2HPO.sub.4, pH=9, Tmax=60° C., time=80 h).

(153) The results are shown in FIG. 4, wherein the black columns refer to the biomorphic hydroxyapatite of the disclosure and the dark gray columns to the hydroxyapatite known in the art.

(154) It is evident the increasing in the number of pores having diameter comprised in the range 200-300 μm in the biomorphic hydroxyapatite of the present disclosure in comparison with known hierarchically organized hydroxyapatite, said pores being the ones with the most appropriate dimensions to promote a physiological vascularization of the biomorphic hydroxyapatite when implanted as bone substitute.

(155) Moreover, the same FIG. 4 shows an increasing number of pores having diameter in the range 0.01-0.1 micron, which clearly indicates that the microstructure of the native wood is preserved in the final product.

Example 3

Preparation of a Hydroxyapatite Derived from Wood Doped with Mg.SUP.2+ and/or Sr.SUP.2+

(156) Steps 1 to 5 of the multi-step method as described in example 1 are followed to yield the calcium carbonate body. Doping with Mg.sup.2+ and/or Sr.sup.2+ ions has been achieved according to each of the following methods:

(157) Method 1

(158) A solution of Sr.sup.2+ (in the form of SrCl) is added to a 1.0 M phosphate-rich solution. The calcium carbonate body prepared according to the multi-step method is then immersed in the combined solution and heated to a temperature of 200° C. under a water vapor pressure of 2 MPa. This yields a Sr.sup.2-doped hydroxyapatite with the morphology of the starting wood piece.

(159) Method 2

(160) The calcium carbonate body is immersed in 1.0 M phosphate-rich solution. Whilst heating to a temperature of 25-90° C. under a water vapor pressure of 0.1 MPa, a solution Sr.sup.2+ is progressively added. This yields a Sr.sup.2-doped hydroxyapatite with the morphology of the starting wood piece.

(161) Method 3

(162) The pure calcium carbonate body (or partially converted in hydroxyapatite by immersion in 1.5 M phosphate-rich solution at room temperature or higher for 24 h) is immersed in an aqueous or organic solution containing Sr.sup.2+ ions for 24 h. It is then removed from the solution and is immersed in 1.5 M phosphate-rich solution. Whilst heating to a temperature of 200° C. under a water vapor pressure in the range 0.5-1.5 MPa. This yields a Sr.sup.2-doped hydroxyapatite with the morphology of the starting wood piece.

(163) Properties of the Scaffold Substituted with Strontium

(164) Hydroxyapatite scaffolds substituted with strontium were developed and were found to exhibit enhanced viability of mesenchymal stem cells (MSCs) when compared to the strontium-free scaffolds (FIG. 5).

(165) The strontium scaffolds also displayed well-spread morphology and increased expression of osteogenesis-relevant genes, such as RUNX2 and ALP (FIG. 6), thus acting as promoters of osteoblastic differentiation. In particular, when compared to the Sr-free scaffold, a significant increase in mRNA level of both the genes (p<0.05) was detected. This increase was particularly high for the scaffold with 2 mol % of Sr.

(166) Enhanced proliferation of pre-osteoblasts over 14 days of investigation was observed when Sr-substituted hydroxyapatite scaffolds were used (FIG. 7). In fact, an increase of strontium in the scaffold yielded a much higher cell viability in the long term. These results imply that new bone formation could be successfully induced and sustained.

(167) The scaffold also demonstrated the possibility of maintaining the osteoblastic phenotype during the two weeks of the investigation (FIG. 8).

(168) The behaviour of cells in contact with the strontium-substituted scaffold was investigated also by observing osteoclast behaviour. A preliminary morphological analysis was carried out to confirm and validate the model of osteoclastogenesis. Osteoclasts grown on the scaffold surface exhibit their typical morphology.

(169) The relative gene expression of the principal marker involved in osteoclast activity and formation (Oscar, Integrin β3 and CatepsinK) was evaluated (FIG. 9). The analysis showed a significant decrease in gene expression over time of all the genes involved in the principal molecular pathways of osteoclasts, thus indicating that the presence of Sr.sup.2+ ions in the scaffold inhibits osteoclast formation and activity.

(170) In conclusion, the substitution of hydroxyapatites with Sr.sup.2+ ions produced a biological effect on bone cells, specifically causing: i) a significant inductive effect on MCSs osteogenic related genes; ii) an inductive effect on osteoblasts proliferation and iii) an inhibitory effect on osteoclasts activity. In the case of implantation in a segmental bone defect, the new scaffold is designed to present a central channel that extends in direction parallel to the main uni-directional porosity so to be exposed to the bone stumps as a guide for new bone marrow development (FIG. 1). The channel size is defined on the basis of the specific detect; however, to maintain adequate strength the channel has a diameter in the range 20-60% in respect to the whole scaffold width.

Example 4

Comparison of Biomorphic Hydroxyapatite of the Disclosure and Hierarchically Structured Hydroxyapatite Known in the Art

(171) A comparison test was made between a biomorphic hydroxyapatite manufactured using the process of the disclosure and a hydroxyapatite scaffold obtained from the same native wood (rattan) according to the teaching of Anna Tampieri et al. in the Journal of Material Chemistry, 2009, 19, 4973-4980. The rattan wood used in the process of the disclosure has a length, measured along a direction in which a dimension of the scaffold is maximum, equal to 2 cm. The rattan wood used in the prior art process has a length, measured along a direction in which a dimension of the scaffold is maximum, equal to 1 cm.

(172) After each step of the two processes, the specific surface area (SSA) and the pore distribution of the intermediate and final scaffolds were analyzed and compared (see FIGS. 10, 16, 19 and 20). In addition, after the carburization step the calcium carbide crystal dimensions of the two intermediate scaffolds were compared (see FIG. 13—bottom pictures).

(173) The crystal lattice structure of the two calcium carbide scaffolds was compared after the carburization step. The comparison is shown in FIG. 14.

(174) FIG. 10 shows a comparison of the pore distribution of the two calcium carbide scaffolds obtained after the respective carburization steps, further compared with the pore distribution of the starting pyrolized wood. The specific surface area of the two calcium carbide scaffolds are also reported in the figure. The results show that only the calcium carbide scaffold obtained after the carburization step of the disclosure preserves the micro and nano-pore distribution of the rattan wood (pores dimension<1 μm). Also the comparison of the two specific surface areas show an improvement for the scaffold according to the present disclosure.

(175) FIGS. 11, 12 and 13—top two pictures show SEM images of the two calcium carbide scaffolds from which the better preservation of the native wood micro and nano-porosity can be clearly seen.

(176) FIG. 13—bottom two pictures show a comparison of the dimension of the carcium carbide crystals. The calcium carbide obtained according to the present disclosure show granes with an average size of about 10 μm, while the granes obtained with the prior art process have an average size of about 100 μm.

(177) FIG. 14 show a comparison of the crystal phase of the two calcium carbide scaffolds, measured with x ray-XRD. The results show that the calcium carbide obtained according to the present disclosure has both a tetragonal and cubic crystal lattice, while the calcium carbide of the prior art has a tetragonal lattice only. The calcium carbide scaffold of the disclosure contains a higher amount of Ca(OH).sub.2 with respect to the prior art scaffold.

(178) FIG. 15 shows SEM images of the calcium oxide scaffold obtained after the respective oxidation steps. The pictures corresponding to the scaffold obtained according to the present disclosure preserves a microporosity between the CaO granes, while in the prior art scaffold the microporosity is completely lost.

(179) FIG. 16 depicts the pore distribution of the two calcium oxide scaffolds. The comparison clearly show that the micro and nano-porosity fraction obtained after the oxidation step is higher in the scaffold according to the present disclosure as well as the specific surface area.

(180) FIGS. 17 and 18 show SEM images of the calcium carbonate obtained after the respective carbonation. The material according to the present disclosure show an extended fine structure, compared to the prior art where large crystals of calcite (up to about 50 μm) characterize the whole structure. The large crystals cause the structure to break or collapse during the phosphatization step.

(181) FIG. 19 shows that the pore distribution of the two calcium carbonate scaffolds obtained after the carbonation step. The comparison of the pore distribution and the specific surface area show a result, similar to the one discussed above for the calcium oxide: the micro and nano-pore structure<1 um is maintained with the present process and an higher SSA is obtained with respect to the prior art.

(182) FIG. 20 shows the comparison between the pore distribution of the final biomorphic hydroxyapatite scaffold (after phosphatization) obtained with the process of the disclosure and the final biomorphic hydroxyapatite scaffold of the prior art. A comparison of the respective SSA is also shown.

(183) The results show that the biomorphic scaffold of the disclosure possess a higher micro and nano-porosity fraction than the prior art scaffold as well as a higher specific surface area.

Example 5

Prior Art Process Applied to a Piece of Rattan Wood Having a Length, Measured Along a Direction in which a Dimension of the Scaffold is Maximum, Equal to 2 cm

(184) A test was made to demostrate that the prior art process (Anna Tampieri et al. in the Journal of Material Chemistry, 2009. 19, 4973-4980) does not allow manufacturing scaffolds having a length, measured along a direction in which a dimension of the scaffold is maximum, equal or greater than 2 cm, i.e. scaffolds of clinical interest for bone regeneration.

(185) To this purpose, a piece of rattan wood has been subjected to the process steps according to the conditions described in Tampieri et al.

(186) FIG. 21 show that even before the phosphatization step the scaffold can break down. FIG. 22 show that even if the scaffold survives the process steps up to phosphatization, after phosphatization the scaffold breaks down.

(187) The test clearly shows that the scale up of a ceramic product is often not a straightforward operation; instead the process conditions need to be changed (sometimes heavily changed) in order to prepare larger products, even when a process for making small ceramic product is known in the art.

Example 6

Comparative Test

(188) An in vitro study was performed with mouse mesenchymal stem cell (mMSCs). Gene expression profiling was analysed in order to test the over expression of specific genes involved in osteogenic differentiation induced by a biomorphic hydroxyapatite scaffold obtained with the process of the present disclosure and a biomorphic hydroxyapatite scaffold obtained with the prior art process.

(189) Sample Description

(190) The two tested scaffolds are defined as follows:

(191) TABLE-US-00001 Disk Number of Sterilization Sample dimension samples method Scaffold of doped Ca/P = 1.65 Ø: 8.00 mm, 5 EtOH + UV the present (disclosure) Mg/Ca = 1.64 L: 4.00 mm irradiation disclosure Sr/Ca = 0.59 Not doped Ca/P = 1.70 Ø: 8.00 mm, 5 EtOH + UV (disclosure) L: 4.00 mm irradiation Scaffold of Not-doped Ca/P = 1.77 Ø: 8.00 mm, 8 25 kGy γ-ray Tampieri (prior art) L: 4.00 mm radiation et al. (GammaRad)

(192) Results

(193) The genes tested, related to both early (Runx2 and ALP) and late stage (OPN) commitment of osteogenic differentiation, seem to be upregulated in the cells grown in all the disclosure scaffolds compared to the prior art scaffolds, with a significant difference for Runx2 and OPN (p≤0.0001). No differences were observed in BMP2 and Col15 gene expression in all the samples tested, probably because BMP2, that is the upstream regulator of the differentiation pathway, after 14 days of dynamic culture, it has already carried out its biological function as suggested by the upregulation of Runx2, Alp and OPN [1]. On the contrary, Col15 is a very late marker related to the production of mineralized bone matrix [2] and probably the time of culture was not sufficient to induce its expression. No differences were observed between the inductive effect of the doped or not-doped disclosure scaffolds (see FIG. 23).

(194) FIG. 23 shows the relative quantification (2.sup.−ΔΔCt) of gene expression with respect to the expression of the not-doped prior art scaffold used as calibrator, after 14 days of mMSCs 3D cultured in dynamic condition with all the tested samples. Average and standard error of three samples were indicated. Statistical analysis was made by two-way ANOVA, followed by Bonferroni's post-hoc test and significant difference is indicated in the graph: ****p≤0.0001.

(195) From the above tests, it is possible to assert that the disclosure scaffolds show a higher inductive power on the expression of osteogenic related genes, with respect to the prior art scaffolds.

REFERENCES

(196) [1] Arch Oral Biol. 2013 January; 58(1):42-9. doi: 10.1016/j.archoralbio.2012.07.010. Epub 2012 Aug. 9. Leader genes in osteogenesis: a theoretical study. Orlando B, Giacomelli L, Ricci M, Barone A, Covani U. [2] J Cell Physiol. 2012 August; 227(8):3151-61. doi: 10.1002/jcp.24001. Extracellular calcium chronically induced human osteoblasts effects: specific modulation of osteocalcin and collagen type XV. Gabusi E, Manlerdini C, Grassi F, Piacentini A, Cattini L, Filardo G, Lambertini E, Piva R, Zini N, Facchini A, Lisignoli G.