Formulation comprising a phosphocalcic cement and a physical and/or covalent hydrogel of polysaccharides, printable and having ductile mechanical properties for bone regeneration/bone repair

Abstract

The present invention relates to the use of a formulation comprising a phosphocalcic cement and a physical and/or ovalent hydrogel of polysaccharides for 3D printing, more particularly for bone regeneration and/or bone repair. The present invention also relates to a kit for 3D printing of bone implants comprising a phosphocalcic cement and a physical and/or covalent hydrogel of polysaccharides as well as to a method to prepare a formulation for 3D printing comprising a step of mixing a phosphocalcic cement and a physical and/or covalent liquid hydrogel precursor of polysaccharides.

Claims

1. A method for 3D printing, in particular for 3D printing of bone implants comprising the use of a formulation comprising a phosphocalcic cement and a physical and/or covalent hydrogel of polysaccharides.

2. The method according to claim 1, wherein said polysaccharides are hyaluronic acid and/or chitosan.

3. The method according to claim 1, wherein said hydrogel is a physical hydrogel.

4. The method according to claim 1, wherein said cement is a tricalcium phosphate cement.

5. The method according to claim 1, wherein the ratio hydrogel/cement is of 2:3 w/w.

6. The method according to claim 4, wherein said hydrogel is a physical hydrogel comprising from 2 to 4% w/v of hyaluronic acid and/or chitosan.

7. The method according to claim 1, wherein said hydrogel is a covalent hydrogel of silated hyaluronic acid and/or silated chitosan.

8. The method according to claim 1, for bone regeneration and/or bone repair.

9. The method according to claim 1 in the context of vertical bone augmentation, complex geometries, large critical size bone defects, or following congenital diseases or to fill bone defects following tumor resections.

10. A kit for 3D printing of bone implants comprising:— (i) phosphocalcic cement; and (ii) a physical and/or covalent hydrogel of polysaccharides.

11. A method to prepare a formulation for 3D printing comprising a step of mixing a phosphocalcic cement and a physical and/or covalent liquid hydrogel precursor of polysaccharides.

12. An implant comprising a formulation as defined in claim 1.

13. Implant according to claim 12, further comprising active molecules such as growth factors and/or cells.

14. A method for bone repair and/or bone regeneration comprising: (i) preparing a formulation comprising a phosphocalcic cement, for example α-TCP powder, and a physical and/or covalent hydrogel of polysaccharides, said hydrogel comprising in particular hyaluronic acid and/or chitosan; (ii) 3D printing of said formulation; and optionally (iii) inserting the implant obtained further to step (ii) in a bone cavity, in particular a complex bone cavity, more particularly a bone cavity with slight undercuts.

15. A method according to claim 14, wherein the cement and the hydrogel are sterilized in a sterile environment or wherein the implant is sterilized before insertion.

Description

FIGURES

[0109] FIG. 1: confocal microscopy image of MSC seeded during 16 days on cover glass coated with collA1 or on HA composite cement and labelled with actin and vinculin.

[0110] FIG. 2: confocal microscopy image of MSC seeded on cement or on HA composite cement during 4 days and labelled with phalloidin.

[0111] FIG. 3: rate of viability of Fibroblast cell line L929 after 4 days of culture on composite HA cement or on cement.

[0112] FIG. 4: scanning microscopy images of HA composite cement (A) and cement (B) after 48 h of immersion in NaCl (0.9%).

[0113] FIG. 5: diffractometer x ray of cement and HA composite cement after 48 h and 21 days of immersion in a saline solution.

[0114] FIG. 6: HA composite HA printed with a pneumatic extrusion process (CellInk) through a 25 g (250 μm) diameter of needle (A, B, C). A disk of 5 mm of diameter, and 1 mm of depth (4 layers) was pretreated with total bone marrow during (40 min) (C). Scanning electronic microscopy image of the materials seeded with Total bone marrow (D).

[0115] FIG. 7: confocal microscopy image of MSC after 16 days seeded on the 3D printed HA composite cement and labelled with phalloidin.

[0116] FIG. 8: microscannotomography of cement (A) and HA composite cement (B)

[0117] FIG. 9: mesenchymal stem cells seeded on cover glass coated with collagen I or on the HA composite cement labelled with the EDU imaging kit.

[0118] FIG. 10: Thixotropic loop of cement, HA composite cement and physical gel of HA.

[0119] FIG. 11: cyclic strain on cement, HA cement composite and gel.

[0120] FIG. 12: absolute viscosity (η.sub.0) of the hyaluronic acid gel and the HA composite cement measured by CROSS equation (A) flow ramp of cement, gel and HA composite cement (B), curve of threshold fluid (C), measure of the threshold deformation (D).

[0121] FIG. 13: absolute viscosity of different molecular weight and concentration of hyaluronic acid gels.

[0122] FIG. 14: flow ramp of different gels (dooted curves) and flaw ramp of different composite formulations (continuous curve) performed with the blending of cement with either chitosan dissolved in acidic solution of acetic acid (AA) or hydrochloric acid (HCL) or with hyaluronic acid dissolved in a glucose solution.

[0123] FIG. 15: cyclic strain of different composite formulations performed with the blending of cement with either chitosan dissolved in acidic solution of acetic acid (AA) or hydrochloric acid (HCL) or with hyaluronic acid dissolved in a glucose solution.

[0124] FIG. 16: young modulus of cement and HA composite cement was measured before and after the sterilization (A, D), compressive strength of cement and HA composite cement was measured before and after the sterilization (B, E), flexural strengths of cement and HA composite cement was measured before and after the sterilization (A, D).

[0125] FIG. 17: deformation of HA composite and cement before and after sterilization.

EXAMPLES

Metabolic Activity

Methodology

[0126] Disks of 2 cm.sup.2 of a composite cement of hyaluronic acid according to the invention were molded in order to fill a culture plate of 24 well. Fibroblast cell line L929 was seeded directly on the disk at a density of 50 000 cells by disk.

[0127] The sample (composite cement of hyaluronic acid L/P=2/3) were compared to the reference (phosphocalcic cement dissolved in the phosphate solution Na.sub.2HPO.sub.4 at the ratio L/P=2/3).

[0128] After 24 h and 3 days, the supernatant of the cell culture media was collected to perform CCK8 test (cell counting kit 8, sigma aldrich) regarding the provider specifications.

Results

[0129] More metabolic activity of fibroblast seeded on the composite cement of hyaluronic acid than on cement reference (the same ratio of physical gel of hyaluronic acid replace by a phosphate buffer) were found.

Cell Adhesion: 2D Culture

Methodology

[0130] Human mesenchymal stem cells were seeded on disks of 2 cm.sup.2 making with the composite cement of hyaluronic acid of the invention at a density of 100 000 cells by disks. After 16 days of culture (FIG. 1) or 4 days (FIG. 2), cells were fixed with 4% paraformaldehyde for 10 min at room temperature then rinsed with BPS buffer. Cells were permeabilized with triton 0.1% for 3-5 min at room temperature. After rinsing with PBS buffer, aspecific site of protein cells were blocked with a solution of bovine serum albumine 1% for 20 min at room temperature.

[0131] Primary antibody of rabbit vinculin polyclonal (Product #PA5-29688 Thermofisher Scientific) at 1/300 in 0.1% BSA and incubated overnight at 4 degree celsius and then labeled with donkey anti-Rabbit IgG (H+L) Superclonal™ secondary antibody, Alexa Fluor® 568 conjugate (life technology) at a dilution of 1:1000 for 45 minutes at room temperature. Then alexa fluor 488 phalloidin (Thermofisher) dissolve 1/1000 in BSA 0.1% were added on cells for 30 min. Confocal macroscope were used for taking photographies.

Results

[0132] Over the cover slips coated with Col1, cells did not express as much as Vinculin than cells on composite cement of hyaluronic acid. This let suppose that hyaluronic acid, the main glycosaminoglycan of the extra cellular matrix improve cell adhesion on the composite cement of hyaluronic acid according to the invention (FIG. 1).

[0133] After 4 days MSC well attached and adhered either on cement or HA composite cement (FIG. 2). Nevertheless it is worth to note that the morphology of cells is different regarding the nature of the material. MSC is better spread on HA composite cement with larger and more numerous filopodes.

Viability

Methodology

[0134] A live and dead labeling (Molecular probes invitrogen) was performed, according to the provider specification, on fibroblast cell line L929 seeded either on cement (L/P=2/3) or on the composite cement of hyaluronic acid of the invention (L/P=2/3). Viability was determined by counting the living cells that metabolize the cleaving of calcein AM (green cells) and the dead cells expressing Ethidium homodimer −1.

Results

[0135] A non-inferiority result was find between cell viability on cement and on the composite cement of hyaluronic acid. The rate of viability is superior to 90% (FIG. 3).

Bioactivity

Methodology

[0136] Composite cement of hyaluronic acid (L/P=2/3) and cement (L/P=2/3) materials were immerged in a solution of NaCl 0.9% during 48 h. After driying in a dessicator the materials was metallized and observed by scanning electronic microscopy imaging.

Results

[0137] Nanoscale needle of apatite can be observed at the surface of HA composite cement (FIG. 4). This microstructure is mimicking the structure of the natural bone and contribute to improve cell adhesion onto the surface of the materials and osseointegration of the material when implanted.

Chemical composition

Methodology

[0138] HA composite cement (L/P=2/3) and cement (L/P=2/3) were molded and immerged in a saline solution (NaCl 0.9%) during 48 h and 21 days. After this different time point of kinetics the materials were dried in a desiccator during one night. The cement and HA composite cement were crushed and analyzed by diffractometer X ray.

Results

[0139] From 48 h it is possible to note the disappearance of the main peaks characterizing the α-TCP while the main peaks characterizing the calcium deficient apatite (CDA) appear (FIG. 5). This chemical composition is close to the mineral phase of the natural bone.

3D Printing: Total Bone Marrow and Mesenchymal Stem Cells Adhesion

Methodology

[0140] HA composite cement paste was loading into a syringe to be extruded by a pneumatic extrusion process through a 25G (250 μm) of diameter needle.

[0141] Implant of 5 mm of diameter and 1 mm of depth was design (4 layers) with a prismatic interconnected porosity.

[0142] The implants was pretreated with total bone marrow during 40 min. After this implant was fixed with glutaraldehyde (4%) and different bath of acetone of increasing concentration (30%, 50%, 70%, 90%, 100%). Then bypassing the critical point of CO.sub.2 was realized. Implant was metallized and observed by scanning electron microscopy imaging (Figure).

[0143] MSC were seeded on 3D printed implants. After 16 days cells were fixed with PFA 4%, permeabilized with Triton (0.1%) and aspecific site blocked with a bovine serum albumin 4%. Then phalloidin (thermofisher 1/1000) was used to labelled actin fibres (FIG. 7).

Results

[0144] It is possible to observe how cells can migrated into the internal structure of the 3D printed implant. Cells attached and adhere on the printed filament.

Intrinsic Closed Mesoporosity

Methodology

[0145] Cement prepared with αTCP powder and phosphate buffer (L/P=2/3) or with αTCP powder and a physical gel of hyaluronic acid (L/P=2/3) were molded. After setting, the bar of the cement was break to analyze the internal microstructure thanks to microscanner imaging.

Results

[0146] Results are shown on FIG. 8.

[0147] A closed porosity can be observed after blending the cement with a visqueous gel of hyaluronic acid.

[0148] On cement only the intrinsec closed microporosity of 100 μm is observed.

Proliferation

Methodology

[0149] A click imaging EDU (thermofisher) was used to study the proliferation. Experience was realized according to the specification of the provider. Only proliferating cells can integrated the EDU into the DNA of the nuclei of cells and catabolism the cleavage of the fluorescent probe allowing a blue fluorescent.

[0150] Proliferation was measured by counting the numbers of cells metabolizing the click reaction by expressing a blue fluorescent compared to the total number of cells.

Results

[0151] After 16 days of culture, cells are able to keep on proliferating only on the composite HA cement (FIG. 9).

Rheology

Methodology

[0152] Rheological behavior of physical gel or composite cement was analyzed using a rheometer (RS300) thanks a flate and striated geometry and plate of 2 mm. Different measurement were realized [0153] thixotropy loop: a shear rate of 0 to 100Pa/s was applied during 60 s then the shear rate has decreased from 100 to 0 Pa/s. The area of the curve can be measured thanks the Rheowin data software. Higher the area of the curve is higher the material is consider as thixotrope. [0154] cyclic strain: deformation of 5% during 1 min was applied. Then the rate of deformation was increased up to 100% during 160 s. Afterward the deformation rate come-back to the starting point. [0155] flow curve: a shear rate of 0-100 Pa/s was applied to folow the evolution of the viscosity.

Results

[0156] The cement (L/P=2/3) is not thixotrope compared to HA gel and the HA composite cement (L/P=2/3). Adding of HA gel to replaced the phosphate buffer make the material thixotrope.

[0157] Results are shown on FIGS. 10, 11 and 12.

[0158] During higher deformation solid modulus of HA gel and HA composite decrease. At the end of the deformation solid modulus of HA gel and HA composite start to be restored. This behavior of the materials is compatible with the characteristics expected for printing.

[0159] Cement (L/P=2/3) is still liquid and start to flow from the shear rate is applied. When phosphate buffer is replaced by physical gel of HA it can be observed the behavior of a newtonien fluid at the beginning of the shear rate applied. The first part of the curve is still linear (the viscosity is independent of the deformation applied) and start to decrease (pseudoplastic region) meaning that the material is a threshold fluid. The rate of the deformation to be applied to start the flowing of the materials was measure by calculating the max of the curve of the shear rate regarding the rate of deformation (C).

[0160] A deformation of 3.375% for gel and 3.680% for the composite (D) were obtained. These rates of deformation are compatible with the strength required to extrude the paste through a 3D printer.

[0161] Cross equation allowed to measure the absolute viscosity of HA gel and HA composite cement. The viscosity of the composite cement is higher than viscosity of physical gel (A).

[0162] The absolute viscosity of lower molecular weight of hyaluronic acid can be similar to that of a higher molecular weight when increasing the concentration of the lower molecular weight of hyaluronic acid (FIG. 13).

[0163] All the gels and composite formulations are rheofluidifiant/pseudoplastic (their viscosity decreased under the shear rate imposed). Cross equation allows to measure the absolute viscosity and displayed an increasing of viscosity of formulations when gel is blending with cement (composite) (FIG. 14).

[0164] All the formulations are self-healing with a beginning of restructuration at the end of the higher rate of deformation (FIG. 15).

Mechanical Tests

Methodology

[0165] HA composite cement and cement L/P=2/3 was molded in a cylindrical mold (12 mm of length and 6 mm od diameter) for compressive tests or molded in rectangular mold (38*38*6 mm) for flexural tests. Thanks to a texture analyzer, the mechanical tests were applied.

[0166] Young modulus was calculated by calculating the slope at the origin of the curve.

[0167] Flexural strength was measured by calculating the breaking point force under 3 points flexion.

[0168] Compressive strength was measured by calculating the breaking point force under compression.

[0169] Deformation was estimated by calculating the lenght of the material at the breaking point force under compression.

Results

[0170] Results are shown on FIGS. 16 and 17.

[0171] The sterilization increases the mechanical properties of the HA composite cement. The young modulus of cement is higher than the composite because it is less elastic/deformable. Nevertheless the compressive strength and flexural strength of the composite is higher than those of the cement because the composite is more deformable (Figure). All the mechanical results are in the range of those found for cancellous natural bone.

CONCLUSION

[0172] The inventors of the present invention have been able to develop a 3D printed formulation that meets all the mechanical, biological and regulatory requirements for bone repair (injectable, printable, cohesive, ductile, biocompatible, biodegradable, sterilizable, certified).