BONE COMPOSITE AND COMPOSITIONS FOR PREPARING SAME

Abstract

Bone Composite and Compositions, particularly multicomponent or multipart compositions, are for the preparation of bone constructs for use in trauma, or cancer patients for example. The multipart compositions are based around combinations of fibrinogen, thrombin, hydrogels and calcium/phosphorous salts. The multipart compositions are capable of being printed to yield bone constructs using a 3D printing process to produce accurate and precise bone constructs of a desired geometry.

Claims

1. A multi-part composition for 3-dimensional printing of a bone composite, the multi-part composition comprising: i) a first part comprising fibrinogen in a pharmaceutically acceptable carrier; ii) a second part comprising thrombin in a pharmaceutically acceptable carrier; and iii) a third part comprising a pharmaceutically acceptable hydrogel mixed with at least one biocompatible inorganic material, the at least one biocompatible inorganic material providing a source of at least one of calcium and phosphorous atoms, wherein the third part has an apparent viscosity selected from the group consisting of: from about 1 to about 300 Pa s at a shear rate of 0.1 s.sup.−1 as measured by a rotational viscometer at 25° C. and 1 atm of pressure, and from about 1 to about 100 Pa s at a shear rate of 1 s.sup.−1 as measured by a rotational viscometer at 25° C. and 1 atm of pressure, and further wherein the third part is a standalone composition, or it is mixed with either the first part or the second part of the multipart composition, such that at least the first and second parts of the multi-part composition do not mix prior to printing by a 3-dimensional printing device.

2. The composition according to claim 1, wherein in said first part the fibrinogen is at a concentration between about 5 to about 200 mg/mL.

3. The composition according to claim 1, wherein in said second part the thrombin is at a concentration about 25 and about 1500 IU/mL.

4. The composition according to claim 1, wherein the third part of the multipart composition has an apparent viscosity selected from the group consisting of: from about 50 to about 250 Pa.Math.s at a shear rate of 0.1 s.sup.−1 as measured by rotational viscometer at room temperature and pressure, and from about 2 to about 50 Pa.Math.s at a shear rate of 1 s.sup.−1 as measured by rotational viscometer at room temperature and pressure.

5. The composition according to claim 1, wherein said third part is a standalone composition such that the three parts of the multipart composition do not mix prior to printing by a 3-dimensional printing device.

6. The composition according to claim 1, wherein said biocompatible inorganic material is selected from the group consisting of bioglass, tricalcium phosphate, single-phase hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, natural bone powder, and combinations thereof.

7. The composition according to claim 1, wherein said biocompatible inorganic material is selected from the group consisting of tricalcium phosphate, single-phase hydroxyapatite, biphasic hydroxyapatite-tricalcium phosphate, and combinations thereof.

8. The composition according to claim 1, wherein the biocompatible inorganic material is beta-tricalcium phosphate.

9. The composition according to claim 8, wherein the beta-tricalcium phosphate has a density of between about 2.95 g/cm.sup.3 and about 3.15 g/cm.sup.3 as determined by helium pycnometry.

10. The composition according to claim 8, wherein the beta-tricalcium phosphate has a density of between about 2.95 g/cm.sup.3 and about 3.10 g/cm.sup.3 as determined by helium pycnometry.

11. The composition according to claim 8, wherein the beta-tricalcium phosphate has a d90 particle size distribution of not more than about 180 μm.

12. The composition according to claim 8, wherein the beta-tricalcium phosphate has a d90 particle size distribution of not more than about 160 μm.

13. The composition according to claim 8, wherein the beta-tricalcium phosphate is characterized by an X-ray powder diffraction pattern comprising unique peaks at °2θ (d value Å); angles of 17.0 (5.2), 21.9 (4.1), 25.8 (3.45), 27.8 (3.2), 29.65 (3.0), 31.0 (2.9), 32.45 (2.75), 34.4 (2.6), 46.9 (1.9), 48.0 (1.9), 48.4 (1.9), and 53.0 (1.7) when obtained with a Cu tube anode with K-alpha radiation.

14. The composition according to claim 1, wherein said biocompatible inorganic material has an average particle size of less than about 150 μm.

15. The composition according to claim 1, wherein said biocompatible inorganic material has an average particle size of less than about 100 μm.

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19. (canceled)

20. (canceled)

21. (canceled)

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24. (canceled)

25. A bone composite obtainable from the multi-part composition according to claim 1.

26. A bone composite according to claim 25, wherein said biocompatible inorganic material is at a concentration of between about 5% w/w to about 60% w/w of the bone composite.

27. A method for preparing a bone composite, the method comprising: i) providing a 3-dimensional printing device with the multi-part composition of claim 1; ii) printing the bone composite according to a determined design; and iii) optionally incubating the bone composite.

28. The method of claim 27, wherein the 3-dimensional printing device prints, i) a first layer comprising either fibrinogen or thrombin; ii) a second layer comprising either fibrinogen or thrombin; wherein if the first layer comprises fibrinogen the second layer comprises thrombin and vice versa, iii) sequentially repeating said i) and ii) n times, wherein n≥1, to generate an alternating layered structure; iv) printing the third part comprising the hydrogel and biocompatible inorganic material on top of the alternating layered structure of said iii); and v) optionally repeating said i)-iv) as necessary to provide the bone composite.

29. The method of claim 28, wherein the fibrinogen is printed first, and the thrombin is printed second as a layer on top of the fibrinogen.

30-43. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0251] Additional features and advantages of the present invention will be made clearer in the appended drawings, in which:

[0252] FIG. 1 illustrates a series of print tests for the three part compositions of the present invention. A structure of defined geometry was printed and the results of the different compositions are outlined in the grid array shown;

[0253] FIG. 2 illustrates a plot of hMSC cell proliferation in the compositions of the present invention as measured by a ATP Cell Proliferation Assay (Promega);

[0254] FIG. 3 depicts a graphic of hMSC cell viability in the compositions of the present invention as measured using the LIVE/DEAD Cell Viability Assay (Life Technologies);

[0255] FIG. 4 shows the results of a hMSC osteogenic cell differentiation assay for the compositions of the present invention; and

[0256] FIG. 5 illustrates a series of print tests for two part compositions of the present invention.

DETAILED EXAMPLES OF THE INVENTION

[0257] It should be readily apparent to one of ordinary skill in the art that the examples disclosed herein below represent generalised examples only, and that other arrangements and methods capable of reproducing the invention are possible and are embraced by the present invention.

EXAMPLE 1

Printing Objects of a Defined Geometry Using a Three-Part Composition of the Present Invention

[0258] Advantageously, the compositions of the present invention allow accurate and precise printing of structures having defined and irregular shapes. Outlined below are a series of tests illustrating the performance of a number of multipart compositions of the present invention. The compositions were loaded into a 3D printing device and a computer design drawing provided the shape of interest. The operation of a 3D printing device is within the normal skill and ability of a person of ordinary skill in the art.

[0259] The components of the three-part compositions of the present invention are outlined below. All the bone constructs derived from the multipart compositions of the invention and illustrated in FIG. 1 contained the same fibrinogen component (first part) and thrombin component (second part), which are outlined qualitatively in Table 1 below.

TABLE-US-00001 TABLE 1 Fibrinogen Component Thrombin Component Human Fibrinogen (80 mg/mL) Human Thrombin (500 IU/mL) Sodium citrate dihydrated Calcium chloride Sodium chloride Human albumin Arginine Sodium chloride Isoleucine Glycine Glutamic acid, monosodium Water for injections Water for injections

[0260] The surgical sealant product VERASEAL, manufactured and marketed by Grifols under Marketing Authorisation No. EU/1/17/1239/001-004 was the source of the fibrinogen and thrombin components utilised in all the experiments outlined in Examples 1-5. The product is also marketed as FIBRIN SEALANT in the United States of America by Grifols under Biologics License Number (BLN) 125640.

[0261] The hydrogel compositions were subsequently prepared using standard methodologies in the concentrations outlined in Table 2. For example, sodium alginate [(75-200 kDa, G/M ratio≤1, viscosity 20-200 mPa*s (PRONOVA UP LVM, DuPont)] 5 g was weighed out in a laminar flow cabinet. MilliQ water (100 mL) was heated to 50° C. and the sodium alginate was added with mixing in the laminar flow cabinet. The water was kept at a constant temperature of 50° C. until the sodium alginate powder was no longer visible (approx. 1 hour). The resulting solution was covered to maintain sterility and cooled to approximately 6° C. to accelerate the gelation process.

HYALUBRIX was utilised directly from the syringe.

TABLE-US-00002 TABLE 2 Polymer Concentration Sodium alginate (75-200 kDa, G/M ratio ≤ 1, 5 g/100 mL water viscosity 140 mPa*s): PRONOVA ® UP LVM, DuPont Hyaluronic acid Sodium (1500-2000 kDa): 1.5 g/100 mL water HYALUBRIX, Fidia Pharma Gelatin (Viscosity 2.75-3.75 mPa*s, 2 g/100 mL water pH 4.5-5.5): Rousselot 250 PS, Rousselot

[0262] Table 3 outlines the constituents of, and their concentrations in the third part of the multipart composition of the present invention. Prior to their incorporation into the hydrogel, the biocompatible inorganic materials (Bioglass, TCP, etc.) in the weights indicated in Table 3, were hydrated with 8 mL of an albumin hydrating solution and manually mixed. The albumin hydrating solution consisted of human serum albumin at a concentration of 20 mg/mL in a saline vehicle. Both the saline and human serum albumin [ALBUTEIN] were obtained from Grifols.

[0263] The resulting suspension was centrifuged at 140 g for 2 minutes. Excess hydrating solution was removed using a micropipette. The hydrated biocompatible inorganic materials were combined with the hydrogels outlined in Table 2 with manual mixing. The resulting compositions are outline in Table 3. The physical characteristics of the biocompatible inorganic materials are listed below for reference:

TABLE-US-00003 β-TCP (Tricalcium <150 μm; helium density 3.016 g/cm.sup.3; phosphate) Calcium:Phosphate (1.4-1.5) β-TCP/Hydroxyapatite 25% β-TCP, 75% Hydroxyapatite; <150 μm; Calcium:Phosphate (1.4-1.5) Bioglass −200 μm; 90% <100 μm

[0264] The #1-9 numbering utilised in Table 3 directly corresponds to the numbering of the bone constructs illustrated in FIG. 1.

TABLE-US-00004 TABLE 3 #1 #2 #3 #4 #5 #6 #7 #8 #9 Alginate — — — — — — 1 mL 1 mL 1 mL Hydrogel Gelatin — — — 1 mL 1 mL 1 mL — — — Hydrogel Hyaluronic 1 mL 1 mL 1 mL — — — — — — acid Hydrogel Bioglass — — 3.143 g — — 3.143 g — — 3.143 g β-TCP 5.250 g — — 5.250 g — — 5.250 g — — HA* + — 4.703 g — — 4.703 g — — 4.703 g — β-TCP * = 75% Hydroxyapatite (HA): 25% β-TCP

[0265] 3D Printing processes for printing the compositions outlined in Tables 1 and 3 all follow a generalised methodology. The fibrinogen and thrombin parts are printed as alternating layers. The number of layers printed is at the operators discretion, but typically ranges between 2-20. Once the desired number of fibrinogen and thrombin layers are printed, the paste composition is printed on top of the layered fibrinogen/thrombin structure.

[0266] Taking third part composition #7 (Alginate & β-TCP) as an example, utilising a 3D printer, the fibrinogen component and thrombin component (Table 1) were printed alternately from separate containers as follows. A first layer of the fibrinogen component with a volume of 0.0177 cm.sup.3 (17.7 μL) was printed onto the printing surface area. A second layer of thrombin with a volume of 0.0177 cm.sup.3 (17.7 μL) is printed on top of the previous layer of fibrinogen. A total of 8 layers of fibrinogen interleaved with 8 layers of thrombin are printed to generate an alternating layered structure.

[0267] Subsequently, 0.149 cm.sup.3 (149 μL) of third part composition #7 outlined in Table 3 was printed on top of the alternating layered structure of fibrinogen and thrombin, again this material was printed from a separate container. The resulting structure comprising the three compositions printed onto one another is termed the first macrolayer. In this macrolayer, the overall ratio of the fibrinogen composition to thrombin composition to composition #7 is approximately 1:1:1. Within the macrolayer, the fibrinogen and thrombin layers react to form fibrin and strengthen the structure.

[0268] Further macrolayers are printed adjacent to, and on top of the first macrolayer in a sequential order according to a design file to build-up the bone constructs outlined in FIG. 1.

[0269] During the printing process, the shear rate in the syringe tip acting on the compositions was determined to be 15 and 40 s.sup.−1.

[0270] Prior to printing an upper macrolayer directly on-top of a lower macrolayer the operator may perform the additional step of printing a base layer on top of the lower macrolayer. The base layer typically consists of a single layer of fibrinogen and a single layer of thrombin. The perimeter of the base layer projects upward to define an open space bounded by an upwardly turned perimeter. The base layer in effect functions and appears like a nest. The upper macrolayer is printed (as discussed supra) into the open space defined in the base layer, and the upwardly turned perimeter provides a nesting function that adds structural support to the upper macrolayer once it is printed.

[0271] For the avoidance of any doubt, the concept of printing a base layer that functions like a nest so as to provide structure and support to further layers printed therein, is a general concept applicable to and combinable with all method steps of the present invention. The concept should be not treated as being isolated to the particular example discussed in the preceding paragraphs. The skilled person should appreciate the general applicability of the base or nest layer to the methods of the invention.

EXAMPLE 2

Compatibility of the Compositions of the Invention With Human Mesenchymal Stem Cells

[0272] The ability of the compositions of the present invention to provide a non-toxic environment to cells, proteins and other bioactive materials is highly advantageous. 3D Printed bone constructs that facilitate cell survival and cell differentiation are highly desirable from a therapeutic perspective.

[0273] Human mesenchymal stem cells (hMSC) were obtained from the bone marrow of human donors. The hMSC were extracted in accordance with GMP practices, and subsequently expanded ex vivo in Dulbecco's Modified Eagle Medium (DMEM) and 10% Human Serum B (hSERB). The cell culture medium was centrifuged, and any excess liquid was removed. The resulting cells formed sediment at the bottom of the centrifuge tube.

[0274] The sediment of hMSC was mixed with compositions #1, #2, #7, and #8 shown in Table 3. The hMSC sediment was mixed with the relevant hydrogel and the resultant mixture was added to the hydrated biocompatible inorganic material. The resultant paste was manually mixed (gently) to avoid any cell damage. The resultant compositions are outlined in Table 4 with the numbering #1′, #2′, #7′ and #8′ for ease of reference.

TABLE-US-00005 TABLE 4 #1′ #2′ #7′ #8′ Alginate — — 1 mL 1 mL Gelatin — — — — Hyaluronic 1 mL 1 mL — — acid Bioglass — — — — β-TCP 5.250 g — 5.250 g — HA* + β- — 4.703 g 4.703 g TCP hMSC 4 × 10.sup.6 cells 4 × 10.sup.6 cells 4 × 10.sup.6 cells 4 × 10.sup.6 cells *= 75% Hydroxyapatite (HA):25% β-TCP

[0275] Compositions #1′, #2′, #7′ and #8′ including the hMSC were mixed with the fibrinogen and thrombin components of Table 1 in-vitro (without 3D printing) in an approximate ratio of 1:1:1 by volume. The resultant mixture was cultured in the standard conditions discussed supra (DMEM+10% hSERB) for 7 days.

[0276] CELLTITER-GLO 3D Cell Viability Assay (Promega) was used to determine the number of viable cells in 3D cell culture based on quantification of ATP, a marker for the presence of metabolically active cells. After cell lysis the luminescent signal obtained was proportional to the concentration of ATP present in the sample and therefore to the number of viable cells present in culture.

[0277] Briefly, an equivalent volume of CELLTITER-GLO 3D Reagent (Promega) was added to the cell culture volume and the contents were mixed vigorously for 5 minutes to induce cell lysis. The plate was incubated at room temperature for an additional 25 minutes to stabilize the luminescent signal to be recorded.

[0278] FIG. 2 illustrates the results of the cell viability assay. From FIG. 2 it is evident that each of compositions #1′, #2′, #7′ and #8′ showed an increase in the ATP concentration at days 3 and 7. Composition #7′ depicted particularly promising results, although all the compositions tested gave positive outcomes. Thus, it can be concluded that the compositions are non-toxic to the hMSC.

[0279] The compositions outlined in Table 4 were also subjected to a LIVE/DEAD Cell Viability Assay (Life Technologies) based on the simultaneous detection of live and dead cells by employing two probes that respectively measure intracellular esterase activity (by calcein AM probe) and plasma membrane integrity (by ethidium homodimer (EthD-1) probe). The nuclear content was stained with Hoechst 33342 fluorescent dye. A fluorescence microscope was used to visualize cell characteristics.

[0280] In FIG. 3, we see images from the cell viability assay captured with a 10× objective at days 0 and 7. The figure shows viable hMSC, dead hMSC, and stained nuclei. At day 0 there is very little to note in relation to any of the compositions. However, at day 7 the large shaded, blotched areas show that a high concentration of cells are viable when mixed with the compositions of the present invention. Thus, from FIG. 3 it is clear that the compositions of the invention are suitable to permit cell adhesion and spatial cell distribution throughout a bone scaffold as well as to promote cell growth.

EXAMPLE 3

Determining the Osteogenic Potential of the Compositions of the Invention

[0281] The osteogenic potency of the compositions outlined in Table 4 was determined by a cell differentiation analysis protocol.

[0282] Compositions #1′, #2′, #7′ and #8′ of Table 4 including the hMSC were mixed with the fibrinogen and thrombin components of Table 1 in-vitro (without 3D printing) in an approximate ratio of 1:1:1 by volume. The volume of the resultant mixture was supplemented with an equal volume of osteogenic differentiation culture media (STEMPRO Basal Media (90%)+STEMPRO Osteo Supplement 10% from GIBCO). The osteogenic differentiation culture media was replaced every 3 days.

[0283] At day 14 the compositions/hMSC were were tested for alkaline phosphatase (ALP) activity using SIGMAFAST BCIP/NBT solution. The compositions before differentiation culture showed no ALP staining (see Day 0, FIG. 4). However, at Day 14 a large number of cells stained positive for ALP activity, as illustrated by the filament type structures stained under Day 14 in FIG. 4. FIG. 4 illustrates a high number of ALP positive cells, for each of compositions #1′, #2′, #7′ and #8′, after 14 days of osteogenic culturing. These findings positively reinforce that the compositions of the present invention support successful differentiation/osteoinduction of hMSC into osteoblasts. Moreover, the cells displayed osteocyte-like morphology upon visual inspection at day 14.

[0284] All of the compositions assayed facilitated the differentiation of the hMSC into osteocytes.

EXAMPLE 4

Printing Objects of a Defined Geometry Using a Two-Part Composition of the Present Invention

[0285] Two part compositions within the scope of the present invention were prepared according to Table 5. Depending upon whether the second composition contained fibrinogen or thrombin, the first composition consisted of the other of fibrinogen or thrombin as outlined in Table 1 supra. The second compositions contained the components outlined in Table 5.

TABLE-US-00006 TABLE 5 #A #B #C #D Alginate — — — 1 mL Hydrogel Hyaluronic 1 mL 1 mL 1 mL — Acid Hydrogel Bioglass — 3.143 g 3.143 g 3.143 g β-TCP 5.250 g — — — Fibrinogen — 5 mL — 5 mL Composition Table 1 Thrombin 5 mL — 5 mL — Composition Table 1

[0286] As illustrated in FIG. 5, the two part compositions of the present invention do not 3D print with the level of control or accuracy of the three part compositions of the present invention. However, promising results were obtained for compositions #A, #B, and #D, and Example 4 serves as a proof of concept study that suitable bone constructs can be 3D printed with a two part composition within the scope of the present invention and that the accuracy can be finessed with further diligence and time.