IONIC-DOPED COMPOSITION METHODS AND USES THEREOF

Abstract

The present disclosure concerns the production of an ionic-doped composition and nanocomposites hierarchically structured incorporating bioactive ions, and its use in regenerative medicine and/or tissue engineering.

Claims

1. A composition for use in regenerative medicine and/or tissue engineering comprising: an enzymatically crosslinked silk fibroin; and a plurality of ionic doped calcium phosphate nanoparticles.

2. (canceled)

3. The composition according to claim 1, comprising 10-20% (w/w) of the enzymatically crosslinked silk fibroin.

4. The composition according to claim 1, wherein the ionic doped calcium phosphate nanoparticles are selected from the group consisting of: α-tricalcium phosphate, β-tricalcium phosphate, hydroxyapatite, and mixtures thereof.

5. The composition according to claim 1, wherein the ionic doped calcium phosphate nanoparticle size is between 1-100 nm.

6. The composition according to claim 1, wherein crosslinked of silk fibroin is obtainable by an enzymatic reaction with horseradish peroxidase and hydrogen peroxide, calcium peroxide or an oxidizer.

7. The composition according to claim 1, wherein the ion of the ionic doped calcium phosphate nanoparticle is selected from the group consisting of: strontium, zinc, manganese, silicon, magnesium, lithium, gallium, and mixtures thereof.

8. The composition according to claim 1, comprising ionic-doped nanoparticles up to 20 wt. %.

9. The composition according to claim 1, wherein the ionic-doped nanoparticles contents up to 10 mol. % of ionic dopants.

10. The composition according to claim 1, further comprising a bioactive molecule, an active ingredient, or both the bioactive molecule and the active ingredient.

11. The composition according to claim 1, wherein the bioactive molecule/active ingredient is selected from the group consisting of: a growth factor, a hemostatic agent, an osteoconductive agent, an antibiotic, an anti-inflammatory agent, an anti-cancer agent, cells, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, and mixtures thereof.

12. (canceled)

13. (canceled)

14. A scaffold comprising the composition of claim 1, wherein the composition has a porosity between 40-80%, and a pore size between 150-350 μm.

15. (canceled)

16. A method for administering a composite adapted for regenerative medicinal use, tissue engineering, or both, comprising the step of: administrating in an injectable form an enzymatically crosslinked silk fibroin and a plurality of ionic doped calcium phosphate nanoparticles combined as the composite.

Description

DESCRIPTION OF THE DRAWINGS

[0047] The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of disclosure.

[0048] FIG. 1—XRD patterns of SF/pure TCP and SF/ZnSrTCP nanocomposites.

[0049] FIG. 2—μ-CT images of the scaffolds: A) 3D acquisition, B) morphometric analysis, and C) 2D porosity of bone and cartilage parts.

[0050] FIG. 3—SEM/EDS analyses of: A) SF/ZnSrTCP nanocomposites, showing the different SF, interface and SF/ZnSrTCP layers, and B) respective EDS elemental analysis of SF layer (left) and SF/ZnSrTCP layer (right).

[0051] FIG. 4—DMA analysis of the scaffolds.

[0052] FIG. 5—Compressive strength A) and compressive modulus B) of the scaffolds.

[0053] FIG. 6—Histological and immunofluorescence analysis of the hOBs and hACs co-cultured in the BdTCP scaffolds for 1, 7 and 14 days. Standard H&E staining was used to evaluate cell distribution and ECM formation. Sirius red (red) staining was used for the visualization of collagen at the ECM, Safranin-O (red) staining was used to detect GAGs formation (scale bar: 200 μm). Representative immunofluorescence images of the osteogenic-related marker OPN (green) and chondrogenic-related marker ACAN (red) in the co-culture system. Nuclei are stained in blue (scale bar: 100 μm). The red arrow indicates a stained area of ECM mineralization. The yellow arrows indicate the stained SF and SF-dTCP layers.

[0054] FIG. 7—Histological and immunofluorescence analysis of the hOBs and hACs co-cultured in the BTCP scaffolds for 1, 7 and 14 days. Standard H&E staining was used to evaluate cell distribution and ECM formation. Sirius red (red) staining was used for the visualization of collagen at the ECM, Safranin-O (red) staining was used to detect GAGs formation (scale bar: 200 μm). Representative immunofluorescence images of the osteogenic-related marker OPN (green) and chondrogenic-related marker ACAN (red) in the co-culture system. Nuclei are stained in blue (scale bar: 100 μm). The red arrow indicates a stained area of ECM mineralization. The yellow arrows indicate the stained SF and SF-TCP layers.

DETAILED DESCRIPTION

[0055] The present disclosure concerns the production ionic-doped composition and nanocomposites hierarchically structured incorporating bioactive ions, and its use in regenerative medicine and/or tissue engineering.

[0056] The present disclosure present disclosure also relates to method for producing hierarchical nanocomposites structures of enzymatically cross-linked silk fibroin hydrogels and calcium phosphates nanopowders (e.g., β- and α-tricalcium phosphate, hydroxyapatite) doped with different ions (e.g. Zn, Sr, Mn, Mg, and Ga).

[0057] In FIG. 1 can be observed that the scaffolds show low intensity peaks located at 20.2° corresponding to the β-sheet crystalline structure (silk-II structure) of native silk fibroin, and the characteristic phases of β-TCP and β-calcium pyrophosphate (CPP) belonging to the TCP and ZnSrTCP powders. These results indicate that the powders were successfully incorporated into the SF, and this fact did not change its structure of β-sheet conformation, which is critical for the maintenance of the mechanical properties and structural stability of the scaffolds.

[0058] In FIG. 2A is possible to observe the porous structure in each layer of the scaffolds with the TCP powder retained only in the composite layers, as confirmed by the blue domain present in the 3D reconstructions scaffolds. In FIG. 2C can be observed that the porosity distribution profile is homogeneous in each scaffold layer; however, a substantial increase of porosity is observed from the interface region until the silk fibroin layers.

[0059] In FIG. 3A can be observed that the scaffolds presented a macro- and micro-porous structure on both layers, presenting macro-pores larger than 500 μm and micro-pores that reach 10 μm. The scaffold layers were well integrated by continuous interface regions of ˜500 μm thickness. From FIG. 3B is possible to see calcium (Ca) and phosphorous (P) ions in the subchondral bone-like layers and interface regions, as well as the presence of Zn and Sr peaks.

[0060] In FIG. 4 is observed that the storage modulus (E′) of the bilayered and monolayered scaffolds increased at lower rates, with increasing testing frequencies (from 0.1 to 10 Hz), ranging from 0.40±0.11 to 0.59±0.21 MPa on bilayered ZnSrTCP scaffolds, 0.26±0.06 to 0.35±0.09 MPa on bilayered TCP scaffolds, and 0.18±0.05 to 0.24±0.09 MPa on SF scaffolds. The loss factor (tan δ) obtained for the bilayered and monolayered control scaffolds were constant when the frequency increased from 0.1 to 10 Hz. All groups of scaffolds presented similar and high loss factor values for the tested frequencies.

[0061] In FIG. 5A and B, the wet compressive modulus of the bilayered ZnSrTCP (0.23±0.06 MPa) and bilayered TCP (0.19±0.09 MPa) scaffolds was higher than that obtained for the corresponding monolayered scaffolds (SF: 0.06±0.04 MPa; SF/ZnSrTCP: 0.17±0.11 MPa; SF/TCP: 0.15±0.08 MPa). The SF scaffolds presented the lowest compressive modulus, as compared to the bilayered (B) and monolayered composite scaffolds.

[0062] In FIGS. 6 and 7, histological and immunofluorescence analysis was performed to assess the phenotypic expression and activity of the osteoblasts and chondrocytes co-cultured in the BdTCP (FIG. 6, wherein B is bilayered and d is ZnSr) and BTCP (FIG. 7) constructs. The results obtained from the H&E staining images, showed that the hOBs and hACs were able to proliferate up to 14 days of culture. At day 7 and day 14, the hACs attached to the macro-pores walls of the SF layers formed self-aggregated clusters, whereas the hOBs spread and filled the inner porous of the SF-dTCP and SF-TCP layers. The newly formed ECM was stained with Sirius red, showing after 14 days of culture a well pronounced collagen matrix deposited in the co-cultured BdTCP and BTCP scaffolds. In the SF layers, the collagen matrix was mainly evidenced in the hACs aggregates. The GAGs deposition on the BdTCP and BTCP constructs was observed at day 14, by the positive staining for safranin-O. An increase of the ECM mineralization was observed up to 14 days of culture in the SF-dTCP and SF-TCP layers, as compared to the lower staining intensity observed on the SF layers. Since the hACs tend to form thick self-aggregated clusters, the staining intensity in these clusters was considerably higher. Up to 14 days of culture, no detectable differences were observed in the type of ECM produced by the hOBs and hACs co-cultured in the BdTCP and BTCP constructs, with that produced on the corresponding monolayered control scaffolds.

Preparation of Silk Fibroin

[0063] In an embodiment, the silk fibroin (SF) is extracted from Bombyx mori cocoons, by removing the sericin with boiling the cocoons in a 0.02 M Na.sub.2CO.sub.3 solution for 1 h, and then rinsing with distilled water. The resulting SF are dissolved in 9.3 M LiBr at 70° C. for 1 h, and then dialyzed against distilled water by using a benzoylated dialysis tubing for 48 h. Afterwards, the SF solution are concentrated by dialysis in a 20 wt. % of PEG solution for 6 h, to yield a solution of 16 wt. %. The tubing will be rinsed in distilled water, and the solution will be collected.

Ionic-Doped Calcium Phosphates Preparation

[0064] In an embodiment, the ionic-doped nanopowders are obtained by aqueous precipitation from calcium nitrate tetrahydrate (Ca(NO.sub.3)4H.sub.2O) and diammonium hydrogen phosphate ((NH.sub.4).sub.2HPO.sub.4) in a medium of controlled pH with the addition of NH.sub.4OH. Ionic-doped nanopowders (0-10 mol. %) are synthesized by adding suitable amounts of the precursor nitrates of the doping elements. The precipitated suspensions are kept for 4 h under constant stirring conditions and matured for further 20 h under rest conditions, at 20-50° C. The resulting precipitates are vacuum filtered, dried at 100° C., and heat treated for 2 h at 1000-1100° C. The nanopowders are grounded under dry conditions in a planetary mill, followed by sieving.

Fabrication of Monolithic and Hierarchically Structured Nanocomposites

[0065] In an embodiment, the SF hydrogels are prepared by using SF solution of 16 wt. % concentration, horseradish peroxidase solution (HRP, 50 μL/mL of SF) and hydrogen peroxide (65 μL/ml of SF). The nanocomposites are obtained by mixing SF solution with varied amount of HRP and H.sub.2O.sub.2 solutions, followed by addition of ionic-doped CaP nanopowders (16-20 wt. %, CaP mass divided by the total mass of SF) and NaCl particles. The gelation process is performed at 37° C. The salt is extracted by immersion in distilled water for 1 day. The nanocomposites are frozen at −80° C. followed by lyophilization up to 4 days.

[0066] All references recited in this document are incorporated herein in their entirety by reference, as if as each and every reference had been incorporated by reference individually.

[0067] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0068] Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term “a cell” or “the cell” also includes the plural forms “cells”. In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0069] Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim.

[0070] Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0071] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

[0072] In addition, it is to be understood that any particular embodiment of the present invention may be explicitly excluded from any one or more of the claims. Where ranges are given, any value within the range may explicitly be excluded from any one or more of the claims. Any embodiment, element, feature, application, or aspect of the compositions and/or methods of the invention, can be excluded from any one or more claims. For purposes of brevity, all of the embodiments in which one or more elements, features, purposes, or aspects is excluded are not set forth explicitly herein.

[0073] The aforementioned embodiments are combinable.

[0074] The following claims further set out particular embodiments of the disclosure.

[0075] The following references should be considered herewith incorporated in their entirety: [0076] 1. Yan L. P., Silva-Correia J., Ribeiro V. P., Miranda-Gonçalves V., Correia C., da Silva Morals A., Sousa R. A., Reis R. M., Oliveira A. L., Oliveira J. M., and Reis R. L., “, Scientific Reports, vol. 6, issue 31037, doi:10.1038/srep31037, 2016. [0077] 2. Yan L. P., Salgado A. J., Oliveira J. M., Oliveira A. L., and Reis R. L., “De novo bone formation on macro/microporous silk and silk/nano-sized calcium phosphate scaffolds”, Journal of Bioactive and Compatible Polymers, vol. 28, issue 5, pp. 439-452, doi:0.1177/0883911513503538, 2013. [0078] 3. Yan L. P., Silva-Correia J., Correia C., Caridade S. G., Fernandes E. M., Sousa R. A., Mano J. F., Oliveira J. M., Oliveira A. L., and Reis R. L., “Bioactive Macro/micro Porous Silk Fibroin/Nano-sized Calcium Phosphate Scaffolds with potential for Bone Tissue Engineering Applications”, Nanomedicine, Nanomedicine, vol. 8, issue 3, pp. 359-378, doi:10.2217/nnm.12.118, 2012. [0079] 4. Yan L. P., Silva-Correia J., Oliveira M. B., Vilela C. A., Pereira H., Sousa R. A., Mano J. F., Oliveira A. L., Oliveira J. M., and Reis R. L., “Bilayered Silk/Silk-NanoCaP Scaffolds for Osteochondral Tissue Engineering: In Vitro and in Vivo Assessment of Biological Performance”, Acta Biomaterialia, vol. 12, issue 2015, pp. 227-241, doi:10.1016/j.actbio.2014.10.021, 2014. [0080] 5. Yan L. P., Oliveira J. M., Oliveira A. L., Caridade S. G., Mano J. F., and Reis R. L., “Macro/micro Porous Silk Fibroin Scaffolds with Potential for Articular Cartilage and Meniscus Tissue Engineering Applications”, Acta Biomaterialia, vol. 8, issue 1, pp. 289-301, 2012. [0081] 6. VAN T. D., TRAN N. Q., NGUYEN D. H., NGUYEN C. K., TRAN D. L., NGUYEN P. T. “Injectable Hydrogel Composite Based Gelatin-PEG and Biphasic Calcium Phosphate Nanoparticles for Bone Regeneration”, Journal of ELECTRONIC MATERIALS, Vol. 45, No. 5, 2016, DOI: 10.1007/s11664-016-4354-3.