BONEGRAFT SUBSTITUTE AND METHOD OF MANUFACTURE

Abstract

The invention concerns a method of making a synthetic, biodegradable, bone graft comprising a three-dimensional porous structure; a formulation of constituents for use in said method; a synthetic, biodegradable bone graft produced by said method; and a method of surgery comprising use of said bone graft.

Claims

1. A method for making a synthetic, biodegradable, bone graft substitute comprising: (a) mixing calcium carbonate with a binding material to provide a powdered mixture, wherein the binding material comprises at least a first component and a second component that react in the presence of a wetting agent and/or a gelling agent to provide a cement or slurry; (b) mixing the powdered mixture of part (a) with a wetting agent and/or a gelling agent to make a cement or slurry; and (c) printing a three-dimensional bone graft substitute (3D-BGS) from the cement or slurry of part (b) onto a platform using a three-dimensional printer.

2.-4. (canceled)

5. The method according to claim 1, wherein the calcium carbonate is provided as a powder.

6. The method according to claim 1, wherein the calcium carbonate is provided in the range 10-60% by weight of the cement, including every 0.1% therebetween.

7. The method according to claim 6, wherein the calcium carbonate is provided in the range 20-50% by weight of the cement, including every 0.1% therebetween, or in the range 25-50% by weight of the cement, including every 0.1% therebetween.

8. (canceled)

9. The method according to claim 1, wherein the calcium carbonate is in the form of nanoparticles or microparticles.

10. The method according to claim 9, wherein the nanoparticles are in the range of about 10-999 nm in diameter, including every 1 nm integer therebetween; and the microparticles are in the range of 1-30 μm, including every 1 μm integer therebetween

11. The method according to claim 1, wherein said first component comprises a first calcium phosphate, and the second component comprises a second calcium phosphate, wherein said first calcium phosphate of the first component has a higher ratio of calcium to phosphate (Ca/P ratio) than the second calcium phosphate of the second component.

12. The method according to claim 11, (a) wherein said first and/or second calcium phosphate is selected from the group comprising: tetracalcium phosphate (Ca4(PO4)2O; TTCP); tricalcium phosphate (2Ca3(PO4)2; TCP); calcium hydrogen phosphate (CaHPO4); calcium dihydrogen phosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP) (b) wherein said first and/or second calcium phosphate is selected from the group comprising: tetracalcium phosphate (Ca4(PO4)2O; TTCP); tricalcium phosphate (2Ca3(PO4)2; TCP); calcium hydrogen phosphate (CaHPO4); calcium dihydrogen phosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP) provided when said first calcium phosphate is TCP said second calcium phosphate is not DCP and vice versa; (c) wherein said first calcium phosphate is tetracalcium phosphate (Ca4(PO4)2O; TTCP) and said second calcium phosphate is selected from the group comprising: tricalcium phosphate (2Ca3(PO4)2; TCP); calcium hydrogen phosphate (CaHPO4); calcium dihydrogenphosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP), or (d) wherein said first calcium phosphate is tetracalcium phosphate (Ca4(PO4)2O; TTCP) and said second calcium phosphate is selected from the group comprising: calcium hydrogen phosphate (CaHPO4); calcium dihydrogenphosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP).

13.-15. (canceled)

16. The method according to claim 1, wherein the binding material comprises the first and second components provided in the range 10-50% by weight of the cement, including every 0.1% therebetween.

17. The method according to claim 16 wherein the first and second components are provided in solid form and are mixed and ground together in the relative amounts wherein the ratio of the first component to the second component is a range of about 0.5 to 5.5.

18. (canceled)

19. The method according to claim 1 wherein part (a) further comprises mixing of an additive wherein said additive is any compound that affects viscosity and so ensures the mixed product has suitable fluidity to flow but not so much that it fails to hold shape when exuded or compressed.

20. (canceled)

21. The method according to claim 19, wherein said additive or said gelling agent is selected from the group comprising: gelatin, collagen, cellulose, self-assembling peptides and a bioink.

22. The method according to claim 19, wherein said additive is provided at a concentration of 10-50% by weight of cement.

23. The method according to claim 1, wherein the mixing in step (a), and/or step (b), with the wetting agent or gelling agent is undertaken at a temperature in the range of 10° C. and 60° C., including every 0.1° C. integer therebetween.

24. The method according to claim 1, wherein the method includes providing a platform onto which the 3D-BGS is exuded, compressed or printed wherein the temperature of the platform is selected between 1° C.-40° C., including every 0.1° C. therebetween.

25. The method according to claim 1, wherein the printing of step (c) comprises depositing the mixture from step (b) layer by layer to produce 3D-BGS wherein each layer is ideally deposited at an angle with respect to the preceding layer to generate a porous structure; is controlled to produce desired pore sizes in the 3D-BGS product wherein said pore sizes are selected according to the intended use; and/or further comprises printing onto a substrate comprising a support material wherein said cement is deposited in a manner such that said support material is incorporated, partially or fully, into the 3D-BGS.

26.-28. (canceled)

29. The method according to claim 1, further comprising incorporating an active agent into the cement, slurry or product prior to or following the exuding, compressing or printing step.

30.-34. (canceled)

35. A mixture for making a three-dimensional bone graft scaffold comprising calcium carbonate and a binding material wherein the binding material comprises a first component and a second component that react in the presence of wetting agent and/or gelling agent to provide a cement or slurry.

36. The mixture according to claim 35 wherein (a) said first and/or second calcium phosphate is selected from the group comprising: tetracalcium phosphate (Ca4(PO4)2O; TTCP); tricalcium phosphate (2Ca3(PO4)2; TCP); calcium hydrogen phosphate (CaHPO4); calcium dihydrogen phosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP):, (b) said first and/or second calcium phosphate is selected from the group comprising: tetracalcium phosphate (Ca4(PO4)2O; TTCP); tricalcium phosphate (2Ca3(PO4)2; TCP); calcium hydrogen phosphate (CaHPO4); calcium dihydrogen phosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP) provided when said first calcium phosphate is TCP said second calcium phosphate is not DCP and vice versa; (c) said first calcium phosphate is tetracalcium phosphate (Ca4(PO4)2O; TTCP) and said second calcium phosphate is selected from the group comprising: tricalcium phosphate (2Ca3(PO4)2; TCP); calcium hydrogen phosphate (CaHPO4); calcium dihydrogenphosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP); or (d) said first calcium phosphate is tetracalcium phosphate (Ca4(PO4)2O; TTCP) and said second calcium phosphate is selected from the group comprising: calcium hydrogen phosphate (CaHPO4); calcium dihydrogenphosphate (Ca(H2PO4)2; CDHP); dicalcium phosphate dehydrate (CaHPO4.2H2O; DCP), or octacalcium phosphate (Ca8H2(PO4)6.5H2O; OCP).

37.-39. (canceled)

40. The mixture according to claim 35, wherein the mixture comprises calcium carbonate in an amount provided in the range 10-60% by weight, including every 0.1% therebetween.

41. A three-dimensional bone graft substitute (3D-BGS) comprising calcium carbonate and a porous scaffold that is cancellous-like in structure and coated, partially or fully, with hydroxyapatite (HA).

42. The three-dimensional bone graft substitute (3D-BGS) according to claim 41 wherein the three-dimensional bone graft substitute (3D-BGS) comprises calcium carbonate in an amount provided in the range 10-60% by weight, including every 0.1% therebetween.

43. A method of treating bone loss comprising inserting into a bone or attaching to a bone a 3D-BGS according to claim 41.

Description

[0102] Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose. The Invention will now be described by way of example only with reference to the Examples below and to the following Figures wherein:

[0103] FIG. 1 shows a schematic representation of a bioprinter for use in the production of a structure according to an illustrative embodiment of the present invention;

[0104] FIG. 2 shows a graph of gelatin viscosity as a function of temperature;

[0105] FIG. 3 shows (a) Bioplotter for 3D printing and (b) an example of 3D-BGS made by the bioplotter;

[0106] FIG. 4 shows a fourier transform infrared (FTIR) spectrum of a 3D-BGS (red) and a standard hydroxyapatite (HA; black);

[0107] FIG. 5 shows a FTIR spectrum of a 3D-BGS with 50% CaCO.sub.3 in powder and soaked in water for 48 hours (black), standard HA (red) TTCP (green) and CaHPO.sub.4 (blue);

[0108] FIG. 6 shows an Alamarblue assay against statistics analysis of 3D-BGS with 35%, 42%, 28% calcium carbonate by weight;

[0109] FIG. 7 shows human mesenchymal stem cells (hMSCs) grown on a 3D-BGS scaffold stained with Live/Dead dye. The blue fluorescence shows all the cells, the green fluorescence shows the living cells, the red fluorescence shows the dead cells.(A) hMSCs grew on the scaffold with 38% percentage by weight CaCO.sub.3. (B) hMSCs grow on the scaffold with 46% percentage by weight CaCO.sub.3. (C) hMSCs grow on the scaffold with 30% percentage by weight CaCO.sub.3;

[0110] FIG. 8. Scanning electron micrograph (SEM) showing the porous structure of the 3D-BGS (A, C), the nano HA crystals on the surface (B) which supports hMSC attachment and growth (D); it is comparable with the porous structure of (E, G), the nano HA crystals on the surface (F) and hMSC attachment and growth (H) on coralline hydroxyapatite/calcium carbonate (CHACC);

[0111] FIG. 9. Micrographies of histology of 3D-BGS and collagen sponge implantation into soft tissue between tibia bone and tibialis anterior muscle. By light microscopy, it illustrated that the gelatin sponge implantation (control) resulted in fibrous tissue between tibia and tibialis anterior muscle (A). After 3 weeks of 3D-BGS implantation, the materials were covered by mainly fibroblast, macrophages; interestingly, there were small patches of bone-like tissue formation (B). The TEM observation confirmed the finding by light microscopy, as in control gelatin sponge implantation, macrophagic responses were observed with fibroblast infiltration for tissue regeneration (C); whereas within the patch of bone-like tissue, typical osteocyte-like cells with canaliculi-like structure and within calcified Lacuna were observed (D);

[0112] FIG. 10. The microCT images illustrated the osteogenic and biodegradation capacity of 3D-BGS on rat femoral bone defects, in comparison with gelatin sponge implantation, at 1 month (A and B), 2 months (C and D) and 3 months (E and F). It is notable that the non-union bone defect in the gelatin sponge implantation (A, C and E), and the callus formation surrounding the 3D-BGS (B), the integration of 3D-BGS with trabecular bone formation in bone marrow cavity (D), and the biodegradation and remodelling of the 3D-BS and callus (F).

[0113] FIG. 11. X-ray diffraction analysis (XRD) analysis of hydroxyapatite (HA), coralline hydroxyapatite/calcium carbonate (CHACC), 3D-BGS and calcium carbonate. In the XRD patterns of the 3D-BGS, the diffraction peaks of calcium carbonate in the 3D-BGS are still visible. More importantly, the diffraction peaks of hydroxyapatite (HA) appear at 2θ values of 29.1°, 32.9°, 34.1°, 39.6° and 49.5° which correspond to (002), (211), (300), (130) and (213) planes, respectively. These diffraction peaks are in a good agreement with the diffraction standard data of pure HA (JCPDS PDF #09-0432)

[0114] FIG. 12. Comparation of FTIR spectra of HA, CHACC, 3D-BGS and calcium carbonate. The reference samples of FTIR spectrum show absorptions at v1—963 cm.sup.−1, v3—1036 and 1095 cm.sup.−1, v4—568 and 600 cm.sup.−1 are due to PO.sub.3.sup.−4 ions, OH.sup.− groups lay at 630 cm.sup.−1. The reference samples of FTIR spectrum show absorptions at v3 peak of 1453.7 cm.sup.−1, v2 peak of 853.8 cm.sup.−1, v1 peak of 1083.8 cm.sup.−1 and v4 peaks of 699.2 and 712.2 cm.sup.−1 corresponding to CO.sub.3.sup.2−. Compared with FTIR spectrum of TTCP (Ca.sub.4(PO.sub.4).sub.2O) the strong peak at 1036 and 1417 cm.sup.−1 was not carried out by TTCP and there is OH.sup.− groups lay at 640 cm.sup.−1 showed up in FTIR of 3D-BGS which indicates the mixture of TTCP and DCDA has converted to HA. Another strong evidence for the presence of HA is the peak showed up at around 961 cm.sup.−1. The v4 absorption peak of CO.sub.3.sup.2− showed in 3D-BGS, which means there is calcium carbonate remaining in 3D-BGS after all the process. The peaks are all shifted form the reference peaks but in a reasonable value.

[0115] FIG. 13. Alamarblue assay against statistics analysis of 3D-BGS, CHACC and cells only demonstrating 3D-BGS is non-toxic human mesenchymal stem cells.

[0116] FIG. 14. Human Mesenchymal stem cells (hMSCs) grown on a 3D-BGS scaffold stained with Live/Dead dye. The blue fluorescence shows all the cells, the green fluorescence shows the living cells, the red fluorescence shows the dead cells.

[0117] FIG. 15. Gross observation of the effect of 3D-BGS on rat femoral intercondylar bone defect model. Over 3 months the control group showed fibre tissue formation at the bone defect sites whereas in 3D-BGS implantation group there were bone formation surrounding the implants and biodegradation over three months. Arrows indicate the scaffolds.

[0118] FIG. 16. The integration of new bone and 3D-BGS materials illustrated by light and backscatter SEM microscopy at 3 months after implantation. [A] the dark coloured area shows implanted 3D-BGS, light grey areas are new trabecular bone formation, and the blue areas are toluidine blue stained cells in bone marrow (containing lipid droplets), blood vessels and bone tissue. The 3D-BGS implants are completely integrated and firmly bond to new trabecular bone tissue. The majority of 3D-BGS was degraded and replaced by new bone formation. [B] High magnification of [A] shows a large particle of biodegrading 3D-BGS within new formed trabecular bone tissue. The scattered small Toluidine blue stained dots are osteocytes; whereas the large Toluidine Blue areas are bone marrow (containing lipid droplets) and new blood vessels. [C] High magnification of the marked area in [B]. Toluidine blue stained dots with canaliculus are osteocytes; whereas the large Toluidine Blue area is new blood vessel. [D] The high electron density crystalline showing by backscatter demonstrated where the 3D-BGS particles are. The white arrows indicated osteocytes lacunae. The osteocytes are closely integrated with 3D-BGS.

[0119] FIG. 17. Micro-illustration of SEM backscatter, Energy-dispersive X-ray spectroscopy (EDS) map and element analysis of cross section of 3D-BGS at 1, 2 and 3 months post implantation. The high crystalline areas are 3D-BGS implants in which the areas reduced over three months (A, B, and C). The EDS mapping showed the calcium (A1, B1 and C1) and phosphate (A2, B2, and C2) composition of the implants and bone tissue.

[0120] FIG. 18. X-ray radiography illustrated that the comparison of 5 bone graft materials implanted in ϕ5 mm bone defects of Bama mini pigs at day 1, 1 month, 3 months and 6 months post implantation. There was non-union of Gelatin Sponge (1) implantation between 1 day and 3 months with bone formation in the defects at 6 months. Two 3D-BGS formula implants (2 and 3) were healed with clear sign of biodegradation (similar density to bone without gaps) in 3 months and the defects recovered at 6 months. Pure non-porous hydroxyapatite (4) was healed but not degraded. Swine allograft (5) was healed but there was still gaps in the bone defect area in 3 months but fully recovered at 6 months.

[0121] Table 1. Parameters of the lab based bioplotter used in printing;

[0122] Table 2. Proportions of gelatin and CaCO.sub.3 in each test at room temperature, gelatin solvent is 35% (percentage by weight) in the cement;

[0123] Table 3. Proportions of gelatin and CaCO.sub.3 in each test at 32° C., gelatin solvent is 42% (percentage by weight) in the cement;

[0124] Table 4. Proportions of gelatin and CaCO.sub.3 in each test at 37° C., gelatin solvent is 35% (percentage by weight) in the cement;

[0125] Table 5. Feasibility test of different percentage by weight gelatin solvent (15% concentration) with a fixed powder component (0.388 g CaHPO.sub.4, 0.612 g TTCP and 1 g CaCO.sub.3);

[0126] Table 6. Weight by percentage of each component in an example 3D-BGS

[0127] Table 7. The proportion of remaining high crystalline implants in backscatter image

Materials and Methods

Materials

[0128] Gelatin from bovine skin was supplied by Sigma-Aldrich; Gelatin powder was supplied by Merk KGaA Germany; hMSC was isolated from bone from patient with informed consent and ethic approval by South-Wales Research Ethic Committee (REC reference: 12/WA/0029); Alamarblue and LIVE/DEAD® Viability/Cytotoxicity Kit was supplied by Thermal Fisher-Life Technology; TTCP (tetracalcium phosphate) was supplied by Shanghai Rebone Biomaterials Co., Ltd; Calcium hydrogen phosphate was supplied by Shanghai Rebone Biomaterials Co., Ltd; Calcium Carbonate (99%) was supplied by Sigma-Aldrich.

Feasibility Study

[0129] Different proportions of calcium Carbonate, TTCP and Calcium hydrogen phosphate were mixed together following the formula:

##STR00001##

and were grinded for 20 minutes. Different concentrations of gelatin solvent was made by mixing gelatin powder and double stilled water and was left in oven overnight by 37° C. The mixed powder and gelatin solvent was well mixed manually in 37° C. environment. The cement (mixture of powders and solvent) was transferred into syringe and cement was pressed to check if it can come out and stand to hold a structure. Cement setting time was also test by mixing TTCP, calcium hydrogen phosphate and double stilled water together, mixtures were leaving in 4% and 37° C. The mixtures were checked if they are agglomerated every 2 minutes.

Viscosity Test

[0130] Gelatin solvent was taken out and loaded on the platform of rotonetic 2 drive (Bohlin Gemini HR). Viscosity and gel transmission temperature was tested using a 20 mm clamp, and testing temperature was 25-37° C.

3D Printing Process

[0131] Calcium Carbonate, TTCP and Calcium hydrogen phosphate was mixed together and was grinded for 20 minutes. Gelatin solvent was made by mixing the powder and deionised water and was left in oven overnight by 37° C. The mixed powder and gelatin solvent was well mixed manually in 37° C. environment. The paste (mixture of powders and solvent) was transferred into syringe. A 2.5 mm diameter metal nozzle was loaded. The scaffold was built by Bio-plotter manufacturer series (EnvisionTec), the parameters are shown in Table 1.

FTIR

[0132] Calcium carbonate, TTCP, Calcium hydrogen phosphate, gelatin, pure hydroxyapatite and 3D printed scaffolds were tested by FT-IR spectrometer (PerkinElmer UATR Two) with a spectral resolution of 4 cm.sup.−1.

Cytotoxicity (hMSC)

[0133] hMSC isolated from the bone of the patients were seeded into T75 flasks and keep them in incubator until about 75% of the flask was covered by cells and cells were cultured into next passage. The media were replaced every 3 days. After 10 days, the cells were seeded on the scaffold which had been washed by PBS twice and immersed in alpha MEM overnight for Alamarblue assay and Live/Dead staining.

Alamarblue Assay

[0134] Wells with scaffolds and cells were washed by PBS twice and were soaked in Alamarblue solvent which made up with 5% Alamarblue and 95% media in incubator for 2 hours. Alamarblue solvent was transferred into the black 96 well plate after 2 hours, the results were shown by the microplate reader (BMH LABTECH, series number: 415-1387).

Live/Dead Staining

[0135] Wells with scaffolds and cells were washed in PBS twice and were soaked in the Live/Dead dye which made up following the ratio PBS 2 ml, Calcein AM 1 μL, EthD-1 2 μL and Hechst 33324 5 μL in incubator for 15 minutes. Cells were washed by PBS twice after stained. Results were shown on confocal microscopy (ZEISS LSM 710).

Scanning Electron Microscopy (SEM) Observation

[0136] The surface structure of coralline hydroxyapatite/calcium carbonate (CHACC) and 3D BGS before and after incorporating hMSCs for 2 weeks were observed by SEM. In brief, at the end of 2 weeks culture of hMSCs on CHACC and 3D BGS, the materials were fixed in 4% glutaraldehyde in 0.1M PBS, dehydrated in a serial of ethanol, the ethanol was replaced by 50% and 100% hexamethlydisilizane, air-dried, sputter-coated with gold and observed by SEM.

Juxtapositional Implantation Between Tibia and Tibialis Anterior Muscle in Rats

[0137] Adult Waster rats (6-8 weeks) were intraperitoneally injected with chloral hydrate at 400 mg/kg for anaesthesia; laid at supine position. The front legs were shaved and a 5-7 mm incision between tibia and tibialis anterior muscle was carefully produced without damaging periosteum on the tibia. The subcutaneous facia was dissected to expose tibia and tibialis anterior muscle, and a 2 mm×2 mm×2 mm (1) clinically applied gelatin sponge; (2) 3D BGS; and (3) 3D BGS containing 10% bioglass were implanted juxtapositionally between tibia and tibialis anterior muscle. The wound was stitched subcutaneously then the fully layer of skin and cleaned with povidone iodine to avoid infection. Analgesia was used for two days. The procedure was approved by local ethical committee at Tongji Medical School, Huazhong University of Science and Technology.

[0138] Three weeks after operation, the rats were euthanized by schedule 1 procedure and the autopsies were harvested and fixed immediately in 4% glutaraldehyde 0.1 M PBS. Then the materials were serially dehydrated in ethanol, embedded in resin. Decalcified semi-thin sections were produced for toluidine blue staining and ultrathin sections for transmission electron microscopy.

Bone Regeneration in Rat Femoral Defect Model

[0139] Adult Waster rats (6-8 weeks) were intraperitoneally injected with chloral hydrate at 400 mg/kg for anaesthesia; laid at supine position. The left front legs were shaved and a 10 mm incision along lateral patellar tendon was carefully produced without damaging periosteum on the femur. The patella was dislocated to expose the femur. A 5 mm×ϕ3.5 mm intercondylar bone defect was created using a dental drill, and groups of 5 mm×ϕ3.5 mm (1) clinically applied gelatin sponge; (2) 3D BGS was implanted in the defect. The patella was relocated and the patellar ligament and subcutaneous ligament were stitched then the full thickness skin. The wounds were cleaned with povidone iodine to avoid infection. Analgesia was used for two days. The procedure was approved by local ethical committee at Tongji Medical School, Huazhong University of Science and Technology. At 1, 2 and 3 months after operation, the rats were euthanized by schedule 1 procedure and the left distal femur containing the implants were harvested and fixed immediately in neutral 10% formalin. The autopsies were scanned by a MicroCT to illustrate the bone defect regeneration; then serially dehydrated in ethanol, embedded in resin. Decalcified semi-thin sections were produced for toluidine blue staining and ultrathin sections for transmission electron microscopy.

MicroCT

[0140] The bone defects were scanned by a MicroCT (Scanco VivaCT40). The data set was collecting using 70 kV voltage, 21 μm layer thickness and 200 ms scanning speed.

Gross Observation

[0141] The gross images were obtained from the sawed samples.

Sectioning and Toluidine Blue Staining

[0142] 10 mm thick sections were cut on a Leica RM2155 motorised microtome with a tungsten knife, and flattened by placing on a drop of isopropanol, overlaying with cling film and rolling with a cylindrical steel rod.

[0143] For light microscopy, sections were stained with 1% toluidine blue in 50 mM Tris buffer pH 7.3 The sections were examined with an Olympus BX51 research light microscope (Olympus Optical Co. (U.K.) Ltd, London, U.K) and digital photomicrographs captured with a Zeiss Axiocam and Axiovision software (Carl Zeiss Vision GmbH, Hallbergmoos, Germany).

TEM

[0144] For TEM, selected areas of blocks were sawn out, re-embedded in LR White resin and 100 nm sections were cut with a glass knife on an Ultracut E ultramicrotome and collected onto 300 mesh nickel grids. All sections were stained with uranyl acetate and lead citrate. For TEM, sections were examined in a Philips CM12 TEM (FEI U. K. Ltd. UK) at 80 kV and images captured with a Megaview III camera and AnalySIS software (Soft Imaging System GmbH, Germany).

Results

Manufacturing of 3D-BGS

[0145] One of the most important elements for a 3D-BGS made by a bioplottor is if the cement can flow out of the nozzle easily. Feasibility was tested at room temperature (27° C.), 32° C. and 37° C. At room temperature, the cement mixture with 10-14% gelatin concentration was liquid-like; cements with 15-19% were more viscous and worked well for the first 4 minutes; cements with 20-25% gelatin were gel-like and could not pass through the nozzle. At 32° C., cements with 10-14% gelatin concentration were fluid; cements with 15-19% were more viscous and worked well for the first 10 minutes; cements with 20-25% gelatin were gel-like and could not pass through the nozzle. At 37° C., cement with 10-14% gelatin concentration was fluid; cements with 15-19% were more viscous and worked well during the entire duration; cements with 20-25% gelatin were sufficiently fluid but found to have limited duration of use only for the first 2 minutes. The results are shown in tables 2, 3 and 4.

[0146] Cement feasibility test of gelatin percentage by weight versus temperature was tested. 33%-36% percentage by weight of gelatin works well at all temperatures. The result is shown in table 5. Cement setting time is 40 minutes at 37° C., 1 hour at room temperature and 2 hours at 4° C.

Viscosity Test

[0147] Gelatin solvent is fluid when its temperature is above 30° C., so viscosity was assessed from 20° C. to 30° C. As shown in FIG. 2 the viscosity changes at about 22° C., thus gelatin with 15% concentration changes from fluid to gel at this temperature (FIG. 2).

3D Printing Process

[0148] Cement was mixed and printed by bioplotter following the parameters showed in table 6. The testing model is shown in FIG. 3.

XRD

[0149] In FIG. 11, the XRD patterns of the 3D-BGS, the diffraction peaks of calcium carbonate in the 3D-BGS are still visible. More importantly, the diffraction peaks of hydroxyapatite (HA) appear at 2θ values of 29.1°, 32.9°, 34.1°, 39.6° and 49.5° which correspond to (002), (211), (300), (130) and (213) planes, respectively. These diffraction peaks are in a good agreement with the diffraction standard data of pure HA (JCPDS PDF #09-0432)

FTIR

[0150] 1 mol TTCP and 1 mol CaHPO.sub.4 should react in water and form hydroxyapatite. FTIR analysis of grafts is shown FIGS. 4 and 5. The red line shows the spectrum of 3D-BGS and the black line shows the spectrum of standard HA sample. In FIG. 5, the black line shows 3D-BGS with 50% CaCO.sub.3 in powder system soaked in water for 48 hours, whilst the red line shows standard HA and green line shows TTCP and blue line shows CaHPO.sub.4. The reference samples of FTIR spectrum show absorptions at v1—963 cm.sup.−1, v3—1036 and 1095 cm.sup.−1, v4—568 and 600 cm.sup.−1 are due to PO.sub.3.sup.−4 ions, OH.sup.− groups lay at 630 cm.sup.−1. The absorptions at 1061 cm.sup.−1, 1217 cm.sup.−1, and 1137 cm.sup.−1 are due to P═O; the absorptions at 1722 cm.sup.−1 is due to HPO.sub.4.sup.−.

[0151] In FIG. 12, the reference samples of FTIR spectrum show absorptions at v1—963 cm.sup.−1, v3—1036 and 1095 cm.sup.−1, v4—568 and 600 cm.sup.−1 are due to PO.sub.3.sup.−4 ions, OH.sup.− groups lay at 630 cm.sup.−1. The reference samples of FTIR spectrum show absorptions at v3 peak of 1453.7 cm.sup.−1, v2 peak of 853.8 cm.sup.−1, v1 peak of 1083.8 cm.sup.−1 and v4 peaks of 699.2 and 712.2 cm.sup.−1 corresponding to CO.sub.3.sup.2−.

[0152] Compared with FTIR spectrum of TTCP (Ca.sub.4(PO.sub.4).sub.2O) the strong peak at 1036 and 1417 cm.sup.−1 was not carried out by TTCP and there is OH.sup.− groups lay at 640 cm.sup.−1 showed up in FTIR of 3D-BGS which indicates the mixture of TTCP and DCDA has converted to HA. Another strong evidence for the presence of HA is the peak showed up at around 961 cm.sup.−1. The v4 absorption peak of CO.sub.3.sup.2− showed in 3D-BGS, which means there is calcium carbonate remaining in 3D-BGS after all the process. The peaks are all shifted form the reference peaks but in a reasonable value.

Cytotoxicity and Life/Dead Staining

[0153] FIG. 6 show the results of relative fluorescence unit on Alamarblue assay and statistics analysis of hMSC seeded on scaffolds with 35%, 42% and 28% CaCO.sub.3 by weight at 7 days and 14 days. There is not much difference between the 3 groups in day 7, but there is a difference between 35% and 42% group in day 14. Compared with day 7 the relative fluorescence units obtained has increased by day 14. FIG. 13 show the results of relative fluorescence unit on Alamarblue assay and statistics analysis of hMSC seeded on 3D-BGS, CHACC and tissue culture plates. Live/Dead staining showed in FIG. 13 indicated that the cell viability was around 92.7±2.8%, which indicates that 3D-BGS is nontoxic to BMSC cells (ISO 10993-5).

[0154] The confocal microscopy of hMSCs stained with Live/Dead dye is shown in FIGS. 7 and 14. The blue fluorescence shows all the cells, the green fluorescence shows the living cells, the red fluorescence shows the dead cells.

[0155] SEM micrographs showed the similar porous structure of 3D-BGS (FIG. 8 A and C) and CHACC (FIG. 8 E and G), the comparable nano HA crystals on the surface of (Fig B and F), and same morphology of hMSC attachment and growth (FIG. 8. D and H).

Juxtapositional Implantation Between Tibia and Tibialis Anterior Muscle in Rats

[0156] The 3D-BGS scaffolds were implanted juxtapositionally between rat tibia and tibialis anterior muscle to observe soft tissue reaction on 3D-BGS in comparison with gelatin sponge. Gelatin is one of additive materials in 3D-BGS.

[0157] By light microscopy, it illustrated that the gelatin sponge implantation resulted in fibrous tissue formation between tibia and tibialis anterior muscle after 3-weeks implantation (FIG. 9A). In contrast, the 3D-BGS implanted at the same time were covered by mainly connective tissue containing blood vessels, fibroblasts and some macrophages; interestingly, there were small patches of bone-like tissue formation (FIG. 9B). The TEM observation confirmed the finding by light microscopy, as in control gelatin sponge implantation, macrophagic responses were observed with fibroblast infiltration for tissue regeneration (FIG. 9C); whereas within the patch of bone-like tissue in the 3D-BGS group, typical osteocyte-like cells with calcified Lacuna and canaliculi-like structure were observed (FIG. 9D).

Bone Regeneration in Rat Femoral Defect Model

[0158] MicroCT scans at 1, 2 and 3 months after gelatin sponge and 3D-BGS implantations are shown in FIG. 10. The (φ3.5 mm bone defects in gelatin sponge implantation group remained non-union (FIG. 10 A, C and E). Even though there were reactive callus formation in the bone marrow cavity at 1-month post operation (FIG. 10A), the calluses were remodelled at 2 (FIG. 10C) and 3 months (FIG. 10E). There was extensive callus formation surrounding 3D-BGS to re-union the bone defects healed at 1 month after implantation, though there were some gaps at the material/bone interface (FIG. 10B). At 2 months after implantation, 3D-BGS completely integrated with host compact bone tissue whereas trabecular bone formed within the porous structure of 3D-BGS (FIG. 10D). At 3 months after implantation, large part of 3D-BGS within the bone marrow cavity was degraded via callus remodelling and the remaining 3D-BGS joined the compact bone of the femur (FIG. 10F).

[0159] Gross observation of the effect of 3D-BGS on rat femoral intercondylar bone defect model are shown FIG. 15. Over 3 months the control group showed fibre tissue formation at the bone defect sites whereas in 3D-BGS implantation group there were bone formation surrounding the implants and biodegradation over three months. Arrows indicate the scaffolds.

[0160] The integration of new bone and 3D-BGS materials illustrated by light and backscatter SEM microscopy at 3 months after implantation are shown in FIG. 16. [A] the dark coloured area shows implanted 3D-BGS, light grey areas are new trabecular bone formation, and the blue areas are toluidine blue stained cells in bone marrow (containing lipid droplets), blood vessels and bone tissue. The 3D-BGS implants are completely integrated and firmly bond to new trabecular bone tissue. The majority of 3D-BGS was degraded and replaced by new bone formation. [B] High magnification of [A] shows a large particle of biodegrading 3D-BGS within new formed trabecular bone tissue. The scattered small Toluidine blue stained dots are osteocytes; whereas the large Toluidine Blue areas are bone marrow (containing lipid droplets) and new blood vessels. [C] High magnification of the marked area in [B]. Toluidine blue stained dots with tails are osteocytes; whereas the large Toluidine Blue area is new blood vessel. [D] The high electron density crystalline showing by backscatter demonstrated where the 3D-BGS particles are. The white arrows indicated osteocytes lacunae. The osteocytes are closely integrated with 3D-BGS.

[0161] Micro-illustration of SEM backscatter, Energy-dispersive X-ray spectroscopy (EDS) map and element analysis of cross section of 3D-BGS at 1, 2 and 3 months post implantation are shown in FIG. 17. The high crystalline areas are 3D-BGS implants in which the areas reduced over three months (A, B, and C). The EDS mapping showed the calcium (A1, B1 and C1) and phosphate (A2, B2, and C2) composition of the implants and bone tissue.

[0162] X-ray radiography illustrated that the comparison of 5 bone graft materials implanted in ϕ5 mm bone defects of Bama mini pigs at day 1, 1 month, 3 months and 6 months post implantation are shown in FIG. 18. There was non-union of Gelatin Sponge (1) implantation between 1 day and 3 months with bone formation in the defects at 6 months. Two 3D-BGS formula implants (2 and 3) were healed with clear sign of biodegradation (similar density to bone without gaps) in 3 months. Pure non-porous hydroxyapatite (4) was healed but not degraded. Swine allograft (5) was healed but there was still gaps in the bone defect area in 3 months but fully recovered at 6 months.

Summary

[0163] We have therefore devised a self-set calcium phosphate/calcium carbonate composition to form a 3D-printed bone graft substitute (3D-BGS) with defined pore size, biocompatibility and controllable biodegradation property. In this study, 3D-BGS was produced and optimised by testing different formulas of compositions to ensure the purged paste had appropriate viscosity and could retain structural integrity after printing. Parameters of printer were adjusted and the biomimetic 3D-BGS with various pore size were fabricated. The products were tested by FTIR spectrum to characterise the composition. The 3D-BGS cytotoxicity was tested by using human mesenchymal stem cells (hMSCs), and further implanted in rat tissue and bone defects to test in vivo biocompatibility, osteogenic capacity and potential biodegradation. Results of FTIR spectrum showed that the 3D-BGS produced by this technique is a mixture of hydroxyapatite (HA) and calcium carbonate (CC). The cytotoxicity test showed that the scaffold is nontoxic and the cells could attach and grow on the material. No infection or adverse tissue reaction were observed after 3D-BGS implantation in comparison to clinically applied gelatin sponge. Interestingly, there were patches of bone-like tissue formation after 3D-BGS implantation juxtapositionally between tibia and tibialis anterior muscle. Callus formation was observed by microCT scanning in 3D-BGS group at 1 month after implantation, followed by full integration with host trabecular and cortical bone at 2 months and biodegradation in bone marrow cavity during the bone remodelling processes at 3 months. However, the same bone defects implanted with gelatin sponge remained non-union until 3 months.

[0164] These results supported the strong osteoconductive potential of 3D-BGS. The new formed bone tissue from host at the site of bone defects was firmly integrated into the implants where there were no observable defects left at 2 and 3 months after implantation.

[0165] The biodegradation of 3D-BGS was confirmed and the surface area of the implants in bone defects was reduced significantly. It is notable that the implants participated into the bone remodelling process, where it is evident that new bone formation was immediately followed by resorption of implants during the callus remodelling, leaving small islands of implants in large area of new bone formation, in particular at 3 months after implantation in murine model.

[0166] The benefit of the porous structure that formed by 3D printing technology is also demonstrated by extensive vascularisation whereby new blood vessels penetrated into the pores to aid bone formation and resorption. The superior results to non-porous HA implants are clearly shown in FIG. 18, while 3D-BGS implanted bone defects were completely healed and the implants were degraded, whilst the non-porous HA implants remained almost unchanged after 6 months post-implantation.

TABLE-US-00001 TABLE 1 Platform Temperature Pressure Speed Temperature 37° C. 3 MPa 50 mm/s 5° C.

TABLE-US-00002 TABLE 2 Gelatin Time concen- 0-2 min 2-4 min 4-10 min >10 min tration Liquid Gel Stand Out Out Out Out    10-14% + − − + + + + 15%-17% + + + + + − − 18%-19% + + + + + − −    20-22% − + + − − − −    22-25% − + + − − − −

TABLE-US-00003 TABLE 3 Gelatin Time concen- 0-2 min 2-4 min 4-10 min >10 min tration Liquid Gel Stand Out Out Out Out    10-14% + − − + + + + 15%-17% + + + + + + − 18%-19% + + + + + + −    20-22% − + + + + − −    22-25% − + + + − − −

TABLE-US-00004 TABLE 4 Gelatin Time concen- 0-2 min 2-4 min 4-10 min >10 min tration Liquid Gel Stand Out Out Out Out    10-14% + − − + + + + 15%-17% + − − + + + + 18%-19% + − − + + + −    20-22% + + + + + − −    22-25% + + + + − − −

TABLE-US-00005 TABLE 5 Gelatin Temperature Percentage RT 32° C. 37° C. 42° C. by weight Out Stand Out Stand Out Stand Out Stand 30% − − − − + + + + 33% + + + + + + + − 36% + + + + + + + − 39% + − + − + − + −

TABLE-US-00006 TABLE 6 Gelatin Component CaCO.sub.3 CaHPO.sub.4 TTCP Solvent (15%) Weight by 28 16.3 25.7 30 percentage

TABLE-US-00007 TABLE 7 Period 1 month 2 months 3 months Percentage (%) 31.9 ± 1.6 12.6 ± 0.7* 8.1 ± 0.6*

ABBREVIATIONS

[0167] 3D-BGS 3D-printed bone graft substitute [0168] CC calcium carbonate [0169] CDHP calcium dihydrogenphosphate, [0170] CHACC coralline hydroxyapatite/calcium carbonate [0171] CPC calcium phosphate cement [0172] DCP dicalcium phosphate dihydrate (CaHPO4.2H2O) [0173] EPS Energy-dispersive X-ray spectroscopy [0174] FTIR Fourier Transform Infrared Spectroscopy [0175] HA hydroxyapatite [0176] hMSCs human Mesenchymal Stem Cells [0177] OCP Octacalcium phosphate [0178] SBF simulated body fluid [0179] SLS selective laser sintering [0180] TCP Tricalcium phosphate [0181] TTCP tetracalcium phosphate Calcium phosphate