Particulate biomaterial containing particles having geodesic forms, method of making the same and using for filling or bone tissue substitution

Abstract

Particulate biomaterial containing particles having geodesic shapes for filling or replacement of bone tissue, and a method of making the particulate biomaterial containing particles having geodesic shapes for filling or replacement of bone tissue, made by rapid prototyping technique (RP), wherein said particles have non-prismatic, semi-spherical geodesic forms.

Claims

1. A particulate biomaterial, comprising calcium salt particles having non-prismatic, semi-spherical, geodesic shapes used as a filler or substitute medium for bone tissue, wherein the calcium salt particles are selected from the group consisting of tricalcium phosphate, calcium phosphate, calcium sulfate, and calcium sulfate hemihydrate.

2. The particulate biomaterial according to claim 1, wherein said particles have non-prismatic, semi-spherical, geodesic forms elaborated by using 3D printing rapid prototyping (RP).

3. The particulate biomaterial according to claim 2, wherein the particle geometry is computationally designed, parameterized according to a thickness of the geodesic, a radius of curvature, an apex angle and major axis length.

4. The particulate biomaterial according to claim 3, wherein the particle geometry allows the growth and cell differentiation of mesenchymal cells (MSC) into osteoblasts within the bone recipient bed.

5. The biomaterial according to claim 1, wherein the particle is a bioabsorbable and biocompatible, microporous, semi-spherical geodesic particle impregnated with growth factors and MSC binding protein, which facilitates cell multiplication on its surface, within its surface and between the collected particles.

6. The biomaterial according to claim 1, wherein the random stacking in the form on which particles occupy the volume of a bone bed is modified in each particular case, but the interstices between the particles remain geodesic.

7. The biomaterial according to claim 1, wherein the volumetric pattern regulating the interstices location, is flexible and adaptable to the bed type.

8. A method of making a particulate biomaterial according to claim 1, comprising the steps of: i) adding water and surfactant as binder to the particles previously printed with powder rapid prototyping (RP) technique system; ii) subjecting the powder in an ammonium phosphate solution with molality of 1-3 mol to ion exchange at temperatures between 70-120° C. during 2-8 hours to obtain calcium phosphate; iii) obtaining the powder formed by bioparticles and forming the particulate filling biomaterial.

9. The method of making a particulate biomaterial according to claim 8, wherein 3D printing powder for rapid prototyping contains growth and chemotactic factors for mesenchymal stem cells that are impregnated in the particle.

10. The method of making the bioparticles of claim 1 for use as bone filler, wherein the particles are made according to the following method: i) generating particles and depositing on culture plates with mesenchymal stem cells from diverse sources and obtaining a cellular development on their surface in all the alternatives; ii) the particles are useful as scaffolds for all the studied stem cell sources; iii) leaving the particles for 12 hours in a culture medium with stem cells, which result in adhesion and incorporation of the cellular elements on their surface and inwards; iv) grafting the particles “loaded” with stem cells onto bone defects in the bone tissue.

11. The biomaterial according to claim 1, wherein the biomaterial particle can be used in traumatology and orthopedics, neurosurgery, maxillofacial surgery, oral surgery.

12. The biomaterial according to claim 1, wherein the biomaterial particles can be used in: Bone graft in osteotomies and vertebral column arthrodesis; Bone graft as filler in hip prosthesis insertion; Bone graft for sealing of craniotomies; Bone graft of residual cavities in jaw or maxilla, after eliminating cystic or tumoral lesion; Bone graft in the recovery of residual alveolar bone ridge height; Graft of residual alveoli after an exodontia.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows an elevated view of the particle.

(2) FIG. 1A shows a top perspective view of the particle.

(3) FIG. 1B shows a section of the particle.

(4) FIG. 2 shows a figure based on a microphotography of a collection of particles.

(5) FIG. 3 shows a 3D computational design of a perspective view of a particle.

(6) FIG. 4 shows a figure based on a microphotography of fresh particles made by 3D printing rapid prototyping (RP) technique.

(7) FIG. 5 shows a figure based on a microphotography of grafted particles (Micro-CT image).

(8) FIG. 6 shows a figure based on a microphotography of bone regeneration in rat cranial vault (Micro-CT image)

BRIEF DESCRIPTION OF THE INVENTION

(9) The present invention relates to a biologically active microporous filler particle, which works as a bone tissue synthetic graft material made by 3D printing rapid prototyping (RP) technique, this through multiple layers of powder of thin thickness and a liquid injection system, so the liquids jetted on the dust layer are absorbed therein, causing agglomeration of the powder. This results in a particle having a non-prismatic, geodesic semi-spherical, complex geometry. The particle has a size no greater than 3,000 microns and not less than 300 microns long in its largest diameter, and is made from a calcium sulfate-rich powder material (bioresorbable material), the particles are then heated during 15 to 25 minutes at a temperature between 250 and 325° C., said particles are subsequently converted to calcium phosphate (biocompatible material) by an ion exchange process, carried out in an aqueous medium rich in PO.sub.4.sup.− ions at a temperature between 70 and 120° C. for a period of time between 2 and 8 hours. The curvature features of the particles geometry and their microporosity are computationally designed and are parameterized according to the thickness (300-500 microns) and length of the major (500-3,000 microns) and minor (300-1,500 microns) axis of the geodesic segment, its curvature radius, apex angle. The geometry of the filler particle will allow cell growth and differentiation into osteoblasts within the bone recipient bed and between and within the collected particles. It is known that the microtopography of the substrate on which the mesenchymal cells (MSC) are present in the bone bed, affects the magnitude of the contractile forces (hundreds of micro-newtons) that cellular machinery must exert to move or remain attached to the bone surface. In this way, the concave and convex curved surfaces by reducing these contractibility stresses would favor the cellular adhesion, while less concave surfaces would favor the cellular locomotion. On the other hand, the magnitude of the curvature radius (2,000-5,000 microns) of the substrate surface will also influence the differentiation cell lines that mesenchymal cell will have. In this way, the flat and convex surfaces will favor the differentiation into osteoblasts. The concave-convex macrogeometry also safeguards the interconnectivity of particles and their mechanical engagement, a process that occurs when the particles are randomly deposited inside the bone bed or defect, thus favoring compactness and volumetric stability of the whole and at the same time allowing the existence of internal channel network so the tissue can be mobilized and regenerated by the cellular fluid. Additionally, these channels would favor the generation of multiple bone neoformation nuclei inside the graft, not depending only on peripheral neovascularization from the recipient bed. The mechanical strength of calcium sulphate hemihydrate (compressive strength of 2 to 9 Mpa, tensile strength between 1 and 4 Mpa, elastic modulus of 3 to 5.5 Gpa) allow the particles to be handled and applied inside the bed without losing their original shape, but on the contrary and thanks to the coupling favored by their concave-convex surfaces, the graft will result into stable three-dimensional reconstructions, even if any wall of the recipient bed is total or partially absent. Finally, the developed particle can be supplemented with bioactive growth factors or can be applied in conjunction with platelet concentrates or stromal cell concentrates to favor the rate of proliferation and differentiation of mesenchymal cells into osteoblasts within the bone bed to be regenerated, in at least 30%, according to results obtained from in vitro and in vivo preliminary comparative experimental animal tests.

(10) Mechanical strength

(11) Calcium Sulfate Hemidrate

(12) TABLE-US-00001 TABLE 1 Compression Tensile Elastic strength strength modulus Mpa Mpa Gpa 2-9 1-4 3-3.5

(13) In summary, it is considered appropriate to protect the design and the method of making a bioabsorbable and biocompatible particle, in the form of a microporous, semi-spherical, geodesic segment impregnated with growth factors, chemotactic factors and MSC binding proteins, which promote and facilitate cell multiplication on its surface; the cell multiplication process, mitosis, which is the division of one cell to produce two others. This is the way in which cells multiply, within and between the collected particles.

DETAILED DESCRIPTION OF THE INVENTION

(14) The present invention relates to a filler particle used as a synthetic graft of bone tissue, dentine tissue.

(15) A filler particle having a complex, non-prismatic, semi-spherical geodesic geometry is made by 3D printing using a binder jetting method.

(16) The filler particle made by powder bed and binder jetting (mostly water and surfactant) 3D printing technique has a major axis length size no greater than 3,000 microns and is made in a material that is a calcium sulphate rich powder (bioreabsorbable material), which is then converted to calcium phosphate (biocompatible material) by an ion exchange process carried out in an aqueous medium rich in PO.sub.4.sup.− ions (ammonium phosphate solution having a molality of 1-3 M, Table 2) at a temperature between 70 and 120° C. and for 2 to 8 hrs.

(17) TABLE-US-00002 TABLE 2 Method of making the biomaterial Drying of Calcium Sulphate Particles, Ionic Exchange, Ammonium Phosphate Solution Ionic Exchange Temperature Particle Mole Hours ° C. Calcium sulfate 1-3 2-8 70-120

(18) In the specific case of the present invention, we have an individual particle with “geodesic” segment geometry, which will form part of the filling. Geodesic geometry is formed by a triangular mesh and, consequently, the triangular structure is self-supported transferring its faces to its vertices called load transfer nodes.

(19) Most commonly this is related to domes or earth geometry due to its shape. “The osculating plane of the geodesic is perpendicular at any point to the plane tangent to the surface. The surface geodesic lines are the “straightest” possible lines (with less curvature) given a point and direction fixed on said surface”

(20) Even though this filler shapes and fills a volumetric space of a bone defect, its stacking does not obey a regular pattern, since it is not a matrix or scaffold or continuous scaffolding but instead it corresponds to a set of deposited particles, which are randomly collected, often with concave-convex coincidences, and which are locked between each other thanks to the irregularity provided by their surface porosity, constituting, as a whole, a stable volumetric scaffolding for bone reconstruction, even without the presence of all the retaining walls of the recipient bed.

(21) In the case of the particle of the present invention the stacking is random. The form in which particles occupy the volume of the bone bed in each case is different, random, often having concave-convex coupling between particles which results in concentric bone neoformation units with interstices between the particles and between these bone formation units.

(22) The location of the interstices is always known or predictable, i.e., especially more rigid, in a regular volumetric pattern, whereas in the case of the present invention this pattern is more flexible and adaptable to the bed type.

(23) The development of this bone filling particulate biomaterial has as main advantages that reparative processes are more predictable and at least 30% faster, recovering the anatomy and function of the damaged structures as soon as possible, allowing the patient to return as soon as possible to normal life.

(24) The object of the present invention is to make a microporous particulate biomaterial having a geodesic segment geometry. The test results indicate that it is possible to print particles in calcium sulphate and then transforming the same into calcium phosphate by ion exchange processes. Initially, the osteoconductive and osteoinductive properties of this particulate biomaterial, manufactured with the RP system, were evaluated by classical in vitro cell development tests, wherein mesenchymal cells, derived from dental pulp, proliferated in intimate contact with and within particles. Subsequently, these mesenchymal cells differentiated into osteoblasts, properties that were evaluated by optical microscopy using calcium deposit staining with Alizarin red and with osteogenic differentiation early markers (expression of RUNX2), taking into account cell morphology. Subsequently, they were evaluated in an in vivo murine experimental model. The images obtained were analyzed with micro-CT, which validated the results of the current scientific literature. It is concluded that the geometry of the particle proposed in the present application favors the attachment of the particles with each other and cell growth between them inside the spaces formed between the topographically curved particle surfaces. This innovation in shape and surface, which allows the attachment of particles, generates concentric units that facilitate cell growth, whereby better results are obtained in large volume grafts, with neovascularization focus in the center of the graft and not just on the walls of the recipient bed. This bone filler particle would be directed to satisfying the surgical needs of orthopedists, neurosurgeons, maxillofacials and general dentists, faced with the challenge of filling defects or bone cavities of a certain volume (greater than a cubic centimeter), with a good prognosis, especially if these have great volume. The present invention also represents an interesting economy in both national and global health area, by enabling the production of large quantities of printed particles by rapid prototyping with a very low production cost.

(25) The biomaterial of the present invention may be applied in: Bone graft in osteotomies and vertebral column arthrodesis (Traumatology and Neurosurgery) Bone graft as filler in hip prosthesis insertion (Traumatology) Bone graft for sealing of craniotomies (Neurosurgery)— Bone graft of residual cavities in jaw or maxilla, after eliminating cystic or tumoral lesion (Oral/Maxillofacial Surgery)— Bone graft in the recovery of residual alveolar bone ridge height (Oral Surgery) Graft of residual alveoli after an exodontia (Oral Surgery)

(26) The elements that are used to make the particulate biomaterial of the present invention are: i. Calcium sulfate, powder. ii. 3D Printing via Rapid Prototyping iii. Stem cells derived from dental pulp

(27) Method of Making the Biomaterial:

(28) The calcium sulphate particles are dried, then subjected to ion exchange in ammonium phosphate solution for 2 to 8 hours and at a temperature between 70 and 120° C., then the resulting particles are washed in distilled water for 2 to 5 minutes, then dried on absorbent paper in contact with atmospheric air for 1 hour.

(29) Particles were generated according to the previously described technique. i) The generated particles were deposited on culture plates with mesenchymal stem cells from various sources (fatty tissue, dental pulp, dental papilla) resulting in cell development on their surface in all the alternatives; ii) The particles were used as a scaffold for all the studied stem cell sources; iii) The particles were left for 12 hours in a culture medium (D-MEM) with stem cells, which resulted in adhesion and incorporation of the cellular elements on their surface and inwards. iv) The particles “loaded” with stem cells were grafted onto bone defects experimentally created in the cranial vault of a rat, obtaining results that demonstrate a bone neoformation at least 30% faster and more effective than on the control side.

Application Example

(30) Method of making the biomaterial:

(31) The calcium sulphate particles are dried in an oven at 300° C. for 10 minutes, then subjected to ion exchange in ammonium phosphate solution for 4 hours at a temperature of 80° C., then the resulting particles are washed in distilled water for 3 minutes, then dried on absorbent paper in contact with atmospheric air for 1 hour.

(32) Particles were generated according to the previously described technique. i) The generated particles were deposited on culture plates with mesenchymal stem cells of dental pulp, resulting in cell development. ii) The particles were used as a scaffold for the studied stem cell source; iii) The particles were left for 12 hours in a culture medium (D-MEM) with stem cells, which resulted in adhesion and incorporation of the cellular elements on their surface and inwards. iv) The particles “loaded” with stem cells were grafted onto bone defects experimentally created in the cranial vault of a rat, obtaining results that demonstrate a bone neoformation at least 30% faster and more effective than on the control side.

(33) Experiment: Experimental model carried out in a rat cranial vault (skull).

(34) The rat surgical protocol was the following, differing only in the bioparticle used in left parietal and right parietal, bioparticles with physiological saline were used in the latter.

(35) Trepanation 1: cranial vault, left parietal

(36) Bioparticles with DP-MSC (mesenchymal cells derived from dental pulp)

(37) Trepanation 2: cranial vault, right parietal Bioparticles with PS (physiological saline)

(38) Details of the protocol are: Specimen in dorsal decubitus, secure to operative table with ad hoc cephalic support. Operative field, with povidone iodine. Cranial approach with antero-posterior oblique linear incision of the dermal and periosteal plane. Exposure of the bone plane of the cranial vault, at both sides of the midline. Trepanation of the left parietal bone, with trephine of 5.0 mm external diameter, with preservation of the dura mater. Trepanation is repeated on the right side of the midline, separated by a 3 mm bone bridge from the left paramedian trepanation. Surgical cleaning of both surgical beds, hemostasis and grafting of: GeoBone® particles impregnated with DP-MSC in the left trepanation GeoBone® particles impregnated with physiological saline in the right side; Both trepanations are covered with a guided regeneration resorbable membrane sheet (for clinical use, available on the market). Close on a plane, with Ethilon 3-0.

(39) Results

(40) TABLE-US-00003 TABLE 3 Test Rat 1: analysis at 15 days: Left hole Thickness Thickness distribution Volume distribution mean range Volume range range in μm in μm in μm.sup.3 in % 17.42-<52.26 34.84 829243057 8.9634 52.26-<87.11 69.69 1475346668 15.9471  87.11-<121.95 104.53 1319098906 14.2582 121.95-<156.79 139.37 1588988614 17.1755 156.79-<191.63 174.21 1215497719 13.1384 191.63-<226.48 209.06 848457444 9.171 226.48-<261.32 243.9 820080004 8.8643 261.32-<296.16 278.74 398685325 4.3094 296.16-<331.00 313.58 291858828 3.1547 331.00-<365.85 348.43 170370390 1.8415 365.85-<400.69 383.27 58076729.1 0.6278 400.69-<435.53 418.11 41654085.2 0.4502 435.53-<470.38 452.95 120838089 1.3061 470.38-<505.22 487.8 73283272 0.7921

(41) Conclusion: This sample shows the presence of segmented structures of larger size compared to the right side of the same rat.

(42) TABLE-US-00004 TABLE 4 Test Rat 1: analysis at 15 days: Right hole Thickness Thickness distribution Volume distribution mean range Volume range range in μm in μm in μm.sup.3 in % 17.42-<52.26 34.84 1287903285 17.2697 52.26-<87.11 69.69 1929851005 25.8777  87.11-<121.95 104.53 1312738171 17.6027 121.95-<156.79 139.37 1262243564 16.9256 156.79-<191.63 174.21 862109494 11.5602 191.63-<226.48 209.06 482146829 6.4652 226.48-<261.32 243.9 258865494 3.4712 261.32-<296.16 278.74 61725030.6 0.8277

(43) Conclusion: There is less presence of large objects and the object size range is around 70 μm. In addition, the large object total volume is much smaller than in the cavity of the left hole.

(44) Results

(45) TABLE-US-00005 TABLE 6 Test Rat 2: analysis at 45 days: Left hole. Thickness Thickness distribution Volume distribution mean range Volume range range in μm in μm in μm.sup.3 in % 17.42-<52.26 34.84 478625425 30.9512 52.26-<87.11 69.69 402349488 26.0187  87.11-<121.95 104.53 186861770 12.0838 121.95-<156.79 139.37 100946915 6.5279 156.79-<191.63 174.21 95146644.3 6.1528 191.63-<226.48 209.06 78872047.4 5.1004 226.48-<261.32 243.9 135960034 8.7921 261.32-<296.16 278.74 67625761.6 4.3731

(46) Conclusion: The same tendency as seen in the previous rat can be observed in the left cavity of this rat, but with a smaller size distribution and with a tendency to have larger sizes than the right one.

(47) TABLE-US-00006 TABLE 7 Test Rat 2: analysis at 45 days: right hole. Thickness Thickness distribution Volume distribution mean range Volume range range in μm in μm in μm.sup.3 in % 17.42-<52.26 34.84 309925852 45.2246 52.26-<87.11 69.69 203004182 29.6225  87.11-<121.95 104.53 82414600.9 12.026 121.95-<156.79 139.37 45984460.4 6.7101 156.79-<191.63 174.21 33442763.3 4.88 191.63-<226.48 209.06 10532487.7 1.5369

(48) Conclusion: The same tendency seen in the previous rat can be observed in the right cavity of this rat, but with a smaller size distribution and with a tendency to have smaller sizes than the left cavity.