MICROARCHITECTURE OF OSTEOCONDUCTIVE BONE SUBSTITUTE

Abstract

The invention relates to an osteoconductive graft comprising a biocompatible material that is interspersed with a network of non-overlapping pores connected by channels, and wherein the pores are characterized by a pore diameter of 0.6 mm to 1.4 mm, the channels are characterized by a channel diameter of 0.5 mm to 1.3 mm, the channel diameter is smaller than the pore diameter and the pores are aligned in one, two or three spatial axes at regularly spaced intervals.

Claims

1. An osteoconductive graft comprising of, particularly essentially consisting of, a biocompatible material, wherein said material is interspersed with a network of non-overlapping spheroidal pores connected by channels, and wherein a) said pores are characterized by a pore diameter of 0.6 mm to 1.4 mm, particularly 0.7 mm to 1.3 mm, more particularly 1.0 mm to 1.2 mm; b) said channels are characterized by a channel diameter of 0.5 mm to 1.3 mm, particularly 0.7 mm to 1.2 mm, wherein said channel diameter does not exceed said pore diameter [particularly wherein the channel diameter is smaller than the pore diameter by a factor of 0.95 or smaller], particularly wherein the channel diameter is at least 0.1mm [more particularly at least 0.2 mm or 0.3 mm] smaller than the pore diameter, and c) said pores are aligned in one, two or three spatial axes at regularly spaced intervals, wherein said intervals are defined by said pore diameter plus 0.3 mm to 1.3 mm

2. An osteoconductive graft comprising of, particularly essentially consisting of, a biocompatible material interspersed with a network of non-overlapping pores connected by channels, and wherein a) said material can be represented as a regular lattice of parallelepipeds (particularly of rectangular parallelepipeds or cuboids, more particularly of cubes) each containing a hollow spheroidal pore, wherein the edges of said parallelepiped (cube) are 0.8 mm to 2.7 mm in length, and the distance of a face of the cube to the nearest point on a wall of said pore ranges between 0.15 and 0.65 mm, particularly 0.3 to 1.0 mm, and b) said pores are connected by channels, wherein the channels are characterized by a channel diameter ranging between 35 and 95% of the pore diameter; of 0.5 mm to 1.3 mm, particularly 0.7 mm to 1.2 mm, wherein said channel diameter does not exceed said pore diameter c) said pores are aligned in one, two or three spatial axes at regularly spaced intervals, wherein said intervals (pore diameter plus channel separating pore from neighbouring pore) are defined by said pore diameter plus 0.3 mm to 1.3 mm

3. The osteoconductive graft material according to claim 1, wherein said pores are regularly aligned to each other in one, two or three spatial axes, particularly along two or three spatial axes that are perpendicular to each other.

4. The osteoconductive graft material according to claim 1, wherein said pores are characterized by a spheroidal shape, wherein each spheroidal pore is characterized by a diameter d(x), d(y)and d(z) in each of the three dimensions of space, and for 90% of pores (particularly for each pore), the relations of said diameters are d(x)=Fd(y);d(x)=Gd(z);d(y)=Hd(z); with F, G and H taking a value of 0.5 to 1.5, particularly with F, G and H taking a value of 0.8 to 1.25, more particularly with said pores being characterized by an essentially spherical shape [d(x)=d(y)=d(z)].

5. The osteoconductive graft material according to claim 1, wherein 90% of said pores are characterized by a pore diameter that differs by 5% from the average of all pore diameters of said material.

6. The osteoconductive graft material according to claim 1, wherein said pore diameter differs by no more than 5% between said pores.

7. The osteoconductive graft material according to claim 1, wherein 90% of said channels are characterized by a channel diameter that differs by 5% from the average of all channel diameters of said material.

8. The osteoconductive graft material according to claim 1, wherein said channel diameter differs by no more than 5% between said channels.

9. The osteoconductive graft material according to claim 1, wherein each of said pores is connected to six other pores by said channels.

10. The osteoconductive graft material according to claim 1, wherein said six channels are oriented perpendicular to each other.

11. The osteoconductive graft material according to claim 1, comprising or essentially consisting of a. a bone substitute material selected from i) a calcium phosphate or a mixture of calcium phosphates, particularly any one of the materials of Table 1, or a mixture thereof, ii) a calcium sulfate, iii) a calcium carbonate, iv) a mixture of any of the foregoing i) to iii), said mixture optionally mixed with Na2O and/or SiO2, and v) magnesium or a magnesium alloy, b. and optionally, a polymer (particularly an acrylate-based polymer, more particularly polymethylmethacrylate).

12. The osteoconductive graft material according to claim 1, wherein said bone substitute material is calcium phosphate, in particular tricalcium phosphate, hydroxyapatite and mixtures thereof.

13. The osteoconductive graft material according to claim 11, wherein the osteoconductive graft material comprises between 50 and 100% (w/w) bone substitute material and between 50 and 0% (w/w) polymer.

14. The osteoconductive graft according to claim 11, wherein the bone substitute material is characterized by a grain size ranging from 0.1 m to 100 m, particularly 0.1 m to 5 m or 3 m to 25 m or 10 m to 50 m or 30 m to 100 m.

15. The osteoconductive graft material according to claim 1, wherein the material is characterized by any one of the following geometrical parameters: TABLE-US-00003 Pore diameter Channel diameter Transparency Porosity [mm] [mm] [%] [%] 0.7 0.5 19.60 41.00 0.6 28.30 54.00 0.9 0.5 13.60 34.68 0.6 19.65 42.19 0.7 26.72 52.02 0.8 34.90 63.00 1.1 0.5 10.00 32.84 0.6 14.40 37.22 0.7 19.63 43.06 0.8 25.64 50.73 0.9 32.45 59.85 1.0 40.07 68.61 1.3 0.5 7.60 33.17 0.6 11.10 35.85 0.7 15.03 39.51 0.8 19.63 44.14 0.9 24.84 50.00 1.0 30.67 57.56 1.1 37.12 65.36 1.2 44.17 72.92

16. A method for making an osteoconductive graft comprising the steps of a. Making a green body by depositing, in a 3-dimensional pattern, an inorganic particulate material selected from any one of the materials specified in claim 11.a, suspended in a photopolymerizable monomer, wherein said pattern is characterized by a network of non-overlapping spherical pores connected by channels, and wherein i) said pores are characterized by a pore diameter of 0.6 mm to 1.4 mm, particularly 0.7 mm to 1.3 mm, more particularly 1.0 mm to 1.2 mm; ii) said channels are characterized by a channel diameter of 0 5 mm to 1.3 mm, particularly 0.7 mm to 1.2 mm, wherein said channel diameter does not exceed said pore diameter [particularly wherein the channel diameter is smaller than the pore diameter by a factor of 0.95 or smaller], particularly wherein the channel diameter is at least 0.1mm [more particularly at least 0.2 mm or 0.3 mm] smaller than the pore diameter, and iii) said pores are aligned in one, two or three spatial axes at regularly spaced intervals, wherein said intervals are defined by said pore diameter plus 0.3 mm to 1.3 mm by subsequently depositing contiguous layers of said material, wherein each layer is between 10 mm and 100 m; b. and sintering said green body for 1-10 days at 500-1700 C.

17. The method according to claim 16, wherein said pores are characterized by a spheroidal shape, wherein each spheroidal pore is characterized by a diameter d(x), d(y)and d(z) in each of the three dimensions of space, and for 90% of pores (particularly for each pore), the relations of said diameters are d(x)=Fd(y); d(x)=Gd(z);d(y)=Hd(z); with F, G and H taking a value of 0.5 to 1.5, particularly with F, G and H taking a value of 0.8 to 1.25, more particularly with said pores being characterized by an essentially spherical shape [d(x)=d(y)=d(z)].

18. A system for performing a method according to claim 16, said system comprising device for added manufacturing and a microprocessor controlling said device for added manufacturing, wherein a. said device for added manufacturing is designed and equipped to deposit subsequent layers of a material as specified in any one of claims 1 to 15, and b. said microprocessor being programmed to deposit said material as a network of non-overlapping pores connected by channels as specified in any one of claims 1 to 15.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0068] FIG. 1 shows a schematic drawing of bone substitutes, design, diverse scaffolds, and in vivo testing of tri-calcium phosphate based scaffolds. Porosogen based scaffolds pore distribution and bottleneck dimension (B) is shown (a) for porosogen based porous scaffolds. Scaffold in dark blue and pores in grey.

[0069] Additively manufactured scaffolds (b) show far more uniform pore distribution and bottleneck dimension. (c) Design of one scaffold from the library. (d) Realization of diverse scaffolds. Scaffolds diverse in pore size and bottleneck dimension are displayed on a five-franc coin with 32 mm in diameter. (e) Screening model. Intra operative view on four scaffolds placed into 4 defects created in the calvarial bone of a rabbit.

[0070] FIG. 2 shows toluidine-blue stained middle sections from untreated and scaffold treated defects: (a) Empty control defect; [0071] (b) Additively manufactured scaffold. Pore diameter of 0.5 mm and bottleneck diameter of 0.5 mm. [0072] (c) Additively manufactured scaffold. Pore diameter of 0.7 mm and bottleneck diameter of 0.5 mm. [0073] (d) Additively manufactured scaffold. Pore diameter of 1.2 mm and bottleneck diameter of 1.0 mm. [0074] (e) Additively manufactured scaffold. Pore diameter of 1.5 mm and bottleneck diameter of 1.0 mm.

[0075] FIG. 3 shows bone bridging in relation to pore and bottleneck dimension. In comparison to the empty control, scaffolds with pore diameters from 0.7 to 1.7 mm and bottlenecks below 1.5 mm perform significantly better. Scaffolds with pores of 1.2 mm and bottlenecks between 0.7 and 1.2 perform significantly better than scaffolds with a pore diameter of 1.5 and bottleneck of 1.2 and pore diameter of 1.7 and bottleneck of 0.7.

[0076] FIG. 4 shows the relationship of microarchitecture and osteoconduction. (a) Bony bridging and pore diameter. Bony bridging in relation to pore diameter of all groups. (b) Bony bridging and bottleneck diameter. Bony bridging in relation to bottleneck diameter of all groups. (c) Bony regenerated area and pore diameter. Bony regenerated area in relation to bottleneck diameter of all groups. (d) Bony regenerated area and bottleneck diameter. Bony regenerated area in relation to bottleneck diameter of all groups. [0077] (e) Schematic drawing of conventional scaffold based on random distributed 0.5 mm diameter pores. Scaffold in dark blue and pores in grey. [0078] (f) Schematic drawing of additively manufactured scaffold based on 1.2 mm pores and bottlenecks of 0.7 mm Scaffold in dark blue and pores in grey.

EXAMPLES

Example 1: Scaffolds with Pores between 0.7 and 1.2 mm Perform Better than Empty Controls

[0079] The inventors employed the computer aided design software tool solidworks (Dassault Systmes SolidWorks Corporation, Waltham, Mass.) to design a library of 20 different scaffolds to fit an established calvarial defect model system (de Wild et al., ibid.) where four non-critical size defects of 6 mm in diameter are created in calvarial bones of rabbits and used for screening purposes (FIG. 1e). Five designs failed mechanically during production or during the in vivo testing and are not reported here. The different designs were fed to a lithography-based additive manufacturing machine (CeraFab7500, Lithoz, Vienna, Austria) to produce them in tri-calcium phosphate supplied as a Lithozbone (Lithoz, Vienna, Austria). After debindering of the scaffolds to eliminate the photoactive binder system and sintering to densify the scaffold to increase the mechanical stability (FIG. 1d) the scaffolds were implanted (FIG. 1e) for four weeks. Histologies (FIG. 2) show that bone formation occurred in and around the scaffolds and that the sintered tri-calcium phosphate based scaffolds are biocompatible. Evaluation of osteoconduction was based on the toluidine-blue stained middle ground sections by the determination of bony bridging and bony regenerated area. Compared to non-treated empty defects, almost all scaffolds induced better bony bridging or bony regeneration (FIG. 2). Only if the pore diameter was 1.5 mm and beyond, bony bridging and bony regeneration was reduced significantly.

[0080] Based on bony bridging as a measure of osteoconduction via the velocity of bone ingrowth into scaffolds or defects it appears that scaffolds with pore diameters between 0.7 and 1.2 mm perform equally well (FIG. 3). Scaffolds with pores of 1.2 mm and bottlenecks from 0.7 to 1.2 mm perform significantly better than scaffolds with pores of 1.5 mm and bottleneck of 1.2 mm. The same applied to scaffolds of pores from 1.7 mm and bottleneck of 0.7 mm. This suggests that pore diameters should be below 1.5 mm to reach a high velocity in defect bridging.

The Optimal Pore Diameter and Bottleneck Dimension for an Osteoconductive Scaffold is between 0.7 and 1.2 mm

[0081] The inventors went on to evaluate bony bridging and bony regenerated area dependency grouped by pore diameter and bottleneck dimension, and found bony bridging to be significantly more complete in scaffolds with pores of 0.7 to 1.2 mm compared to a pore diameter of 1.5 mm (FIG. 4a) or 1.7 mm. For bony regenerated area, a pore diameter of 1.0 and 1.2 appeared significantly superior to a pore diameter of 1.5 mm (FIG. 4c). Bony bridging and bony regenerated area of scaffolds with 0.5 mm pores were always in the range of scaffolds with 1.5 mm diameter pores and below scaffolds with pores of 0.7, 1.0, and 1.2 mm in diameter. Therefore, optimal pore diameter for both measures of osteoconductivity lays between 1.0 and 1.2 mm. If the bottleneck between pores is 0.7 or 1.0 mm, bony bridging is significantly higher than for bottlenecks of 1.5 mm in diameter (FIG. 4b). For bony regenerated area, no significant difference between the bottleneck dimensions was observed.

Discussion

[0082] The inventors produced a library of tri-calcium phosphate based scaffolds and tested in vivo the influence of pore and bottleneck dimensions on osteoconduction determined by bony bridging of the defect and percentage of bony regenerated area. They chose an in vivo model system, since osteoconduction is a complex phenomenon, not well understood, originally described by histologies and due to the fact that in vivo and in vitro data are contradictory to each other (Zadpoor (2015) Biomater Sci 3, 231-245). The complexity of osteoconduction might arise from an interplay between, materials, surfaces, and microarchitectures, affecting individual osteogenic cells but also overall vascularization and directional bone tissue growth. Due to shortcomings of present in vitro systems, an in vivo approach was believed mandatory. The focus of the study on which the present specification is based, was on the microarchitecture of the scaffold.

[0083] The results presented herein suggest that the optimal pore size for an osteoconductive bone tissue engineering scaffolds is in the range of 1.0 to 1.2 mm and the bottleneck between pores between 0.7 and 1.0 mm. These values double or even quadruple the size of optimal pores for osteoconduction, and will have a deep impact on the microarchitecture of future, now truly osteoconductive scaffolds in terms of defect bridging, directional bone in-growth into scaffolds (FIG. 4 e, f). Pores and bottlenecks of 1.5 mm reduce defect bridging significantly and might compromise bone regeneration to a point where non-unions are induced. Clinically, the major complication in bone regeneration and the cause of numerous costly revision surgeries are non-unions. Therefore, the development of truly osteoconductive bone substitutes might reduce the number of failed bone regeneration procedures by non-unions significantly. Pores of 0.5 mm appear to perform worse than pores between 0.7 and 1.2 mm in diameter. Since pores of 0.5 mm in this overall scaffold design are right at the limit of our additive manufacturing machine, a final conclusion should not been drawn based on this system. Here, we clearly show that pores and bottleneck dimension below 1.5 mm and preferentially between 1.0 and 1.2 mm are optimal for osteoconduction and set new margins for osteoconductive scaffold microarchitectures.

[0084] For osteoconduction, the inventors found the optimal pore size in the range of 1.0 to 1.2 mm, and certainly below 1.5 mm. One can speculate that these pore and bottleneck dimensions reflect a balance of the positive interactions of directionally growing bone tissue during the course of bridging a defect with the surface of the scaffold, and the negative interactions due to bone growth restrictions by the scaffold. That the positive effects outweigh the negative one is evident by the fact that compared to empty untreated defects, all implants with pores between 0.7 and 1.7 mm and bottlenecks between 0.5 and 1.2 mm performed significantly better (FIG. 3).

[0085] In certain embodiments, the scaffolds presented herein consist of tri-calcium phosphate, which is advantageously used for scaffolds in bone tissue engineering, because it degrades faster than hydroxyapatite, the natural component of bone, but remains biodegradable even after sintering at temperatures above 1100 C. Biodegradation, however, was not an issue in the test system employed herein, since even tri-calcium phosphate degrades in months and not during a 4-week period, as was studied in the model system.

[0086] Additive manufacturing of free form scaffolds not restricted by the filament dimension of extrusion based additive manufacturing methodologies proved useful to study the microarchitecture of osteoconductive bone substitutes. Previously, porous bone substitutes were mainly produced with the help of porosogens resulting in an at random pore distribution, uncontrollable bottleneck dimensions, and a pore diameter between 0.3 and 0.5 mm (FIG. 4e). Based on our results the pore diameter of osteoconductive bone substitutes was increased to 1.0-1.2 mm and the bottleneck dimension to 0.7-1.0 mm (FIG. 4f) to optimize bone bridging and extent of bony regenerated area. Both will most likely diminish the formation of non-unions. Moreover, additive manufacturing is key in the realization of reproducible osteoconductive microarchitectures, where the microarchitecture can be adjusted according mechanical needs. Combined with the patient specific bone defect dimension, additive manufacturing will be central to realize a personalized treatment of any surgical procedure, where predictable bone regeneration with a low chance for the occurrence of non-unions is needed.

Materials and Methods

Design and Fabrication of Scaffolds

[0087] The inventors used the computer aided design software tool solidworks (Dassault Systmes SolidWorks Corporation, Waltham, Mass.) to design a library of 20 different stepped scaffolds with a diameter of 6 mm in the lower 3 levels and 7.5 mm in the upper level as previously reported (24) and illustrated in FIG. 1c,d. The fabrication of tri-calcium phosphate based scaffolds was performed by a CeraFab 7500 (Lithoz, Vienna, Austria), with LithaBone TCP 200 (Ca3(PO4)2) as photosensitive slurry, consisting of tri-calcium phosphate powder of particle size in the range of 5-30 m, acrylate-based monomer, organic solvent (polypropyleneglycol), light absorber and photoinitiator at 43% of solid loading. The CeraFab 7500 (Lithoz, Vienna, Austria) was used to solidify the slurry in a layer by layer fashion resulting in a scaffold green part with a resolution, of 25 m in layer thickness and 50 m in the x/y-plane. After production, the green parts were removed from the building platform, cleaned from undetached slurry, and underwent a thermal treatment process to remove the solvent, to decompose the polymeric binder and to sinter the samples. The program for thermal treatment was provided by the manufacturer, and included a final sintering step of 3 h at 1100 C.

Animal Experiments

[0088] All animal procedures were approved by the Animal Ethics Committee of the local authorities (Canton Zurich, 108/2012 and 115/2015) and performed in accordance with the ethics criteria contained in the bylaws of the Institutional Animal Care and Use Committee. After the acclimatization period, bone regeneration was determined at the calvarial bone of 50 rabbits (female, 26 week old, New Zealand white rabbit) as described earlier (Karfeld-Sulzer et al. (2014) J Tissue Eng Regen Med). Sample size was determined by power analysis.

Histomorphometry

[0089] The evaluation of all implants was performed from the middle section using image analysis software (Image-Pro Plus; Media Cybernetic, Silver Springs, Md.). The area of interest (A01) was defined by the 6 mm defect dimension and the height of the implant, corrected for differences in height between groups of different pore dimension. We determined the area of new bone in the A01 as percent of bone and bony integrated scaffold in the AOI (bony area, %). For the empty control value, the average corrected area occupied by all scaffolds was taken into account.

Bone Bridging The determination of bone bridging was performed as reported earlier (Kruse et al. (2011) Clin Oral Implants Res 22, 506-511; Schmidlin et al. (2013) Clin Oral Implants Res 24, 149-157).

[0090] In brief, areas with bone tissue were projected onto the x-axis. Next, the stretches of the x-axis where bone formation had occurred at any level were summed up and related to the defect width of 6 mm. Bone bridging is given in percentage of the defect width (6mm) where bone formation had occurred.

Statistical Analysis

[0091] The primary analysis unit was the animal. For all parameters tested, treatment modalities were compared with a Kruskal-Wallis test, followed by Mann-Whitney signed rank test for independent data (IBM SPSS v.23). Significance was set at P0.05. Values are reported as either meanstandard error of the mean or displayed in box-plots ranging from the 25th (lower quartile) to the 75th (upper quartile) percentile including the median and whiskers showing the minimum and maximum values.