Functionalized materials for bone implants and dentistry composites

Abstract

The present invention concerns the field of bone implants suitable for replacing a tooth or a bone. Specifically, the invention relates to a coating for metallic dental or orthopedic implants useful in the biomedical and dentistry fields. Further aspects of the invention relate to a process for the preparation of a coated metallic implant and coated metallic implants obtainable by the process.

Description

[0001] “Functionalized materials for bone implants and dentistry composites”

[0002] FIELD OF THE INVENTION

[0003] The present invention concerns the field of bone implants suitable for replacing a tooth or a bone.

[0004] Specifically, the invention relates to a coating for metallic dental or orthopedic implants useful in the biomedical and dentistry fields.

[0005] Further aspects of the invention relate to a process for the preparation of a coated metallic implant and coated metallic implants obtainable by the process.

[0006] STATE OF THE ART

[0007] Bone tissue is continuously remodeled through bone-forming osteoblasts and boneresorbing osteoclasts activity. Even though the excellent repair ability and regeneration capacity of bone, large-scale bone defects require new materials and tools for producing and stimulating bone regeneration.

[0008] Technologies associated with implants are being used for installing a replacement tooth or bone in the human body. The term "implant" originally means an alternative that restores the lost tissue of the human body.

[0009] An implant is prepared in such a manner that an artificial device, which replaces the lost tooth or bone, is made of a material that is not rejected by the body. The implant is required to restore the original function of the tooth or bone.

[0010] Titanium (Ti) is an excellent choice for bone implants due to its ability to integrate with bone tissue. Several time-consuming and expensive techniques have been used to enhance the surface roughness of the implants and obtain porous structures with larger specific surface area that can promote adsorption of proteins, cell adhesion and growth. The modifications of titanium substrates with micro- and nanoscale features remain a challenge to be addressed. In this context, the role of nanomaterials became pivotal for the clinical success and the development of Ti implants with enhanced integration for bone tissue replacement.

[0011] To repair bone defects autogenous bone and other grafting materials are also used, but many drawbacks such as lack of fixture absorption and difficulties of bone donation in the desired amount can impair the desired effect. In some cases, membranes are used to enhance bone absorption, but a disadvantage of some membranes is their swelling when the bone defect is closed, which causes tension in the remaining tissues.

[0012] Object of the present invention is the development of materials that can be used to texturize smooth implant surfaces and promote osteogenesis.

[0013] SUMMARY OF THE INVENTION

[0014] The inventors found that through the application of particular microalgae, metallic materials used for surgery of humans and animals can be coated in such a way as to promote regeneration of the surrounding tissue, avoiding long and difficult healing processes and risk of infection.

[0015] In the present invention CBD was used to in vivo functionalize microalgae, thereby providing the effect of CBD, in combination with the nanostructured cell walls, on proliferation and osteogenic differentiation of human mesenchymal stem cells (hMSCs) for bone tissue engineering applications.

[0016] A first aspect of the present invention relates to a coating for a metallic dental or orthopedic implant, said coating comprising one or more microalgae, said microalgae being functionalized with Cannabidiol (CBD), wherein said coating promotes cell adhesion, cell proliferation and / or cell differentiation.

[0017] In a second aspect, the use of the coating for a metallic implant is herein described for biomedical and dentistry applications.

[0018] In a third embodiment, the invention relates to a metallic implant having an outer layer, said outer layer being the coating comprising one or more microalgae functionalized with CBD.

[0019] In a fourth aspect, the invention relates to a process for the preparation of a coated metallic implant, comprising the steps of : a. preparing a culture of living microalgae; b. adding Cannabidiol to the culture of living microalgae of step a. to obtain a mixture; c. allowing the Cannabidiol of the mixture of step b. to be adsorbed by the living microalgae, to obtain a culture of microalgae functionalized with Cannabidiol; d. immersing a metallic implant in the culture of microalgae functionalized with Cannabidiol of step c.; e. obtaining a coated metallic implant.

[0020] A fifth aspect of the present invention relates to a coated metallic implant obtainable by the process as herein described. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The invention is described in detail below with reference to the Figures, specifically:

[0022] Figure 1. Number of microalgae cells grown in CBD-supplemented (0.01 mg / mL, 0.05 mg / mL and 0.1 mg / mL) growth medium over time (10 days).

[0023] Figure 2. GC-MS analysis of CBD-supplemented growth media after soaking, adsorption and uptake processes by microalgae.

[0024] Figure 3. ATR-FTIR analysis of CBD (*), bare microalgae (°) and functionalized microalgae with 0.01 mg / mL (**), 0.05 mg / mL (***) and 0.1 mg / mL (****) of CBD.

[0025] Figure 4. bidimensional fluorescence microscopy of smooth Ti (left) and Titanium CBD microalgae scaffold (right)

[0026] Figure 5. SEM image of CBD-functionalized microalgae on Ti scaffold.

[0027] Figure 6. Relative changes in gene expressions of RUNX2, SP7 and COL1A 1 of hMSCs on Ti@CBD microalgae, normalized to GAPDH.

[0028] DETAILED DESCRIPTION OF THE INVENTION

[0029] Naturally available and biocompatible materials that stimulate bone regeneration are required for bone implants.

[0030] The present invention concerns a coating for a metallic dental or orthopedic implant, said coating comprising one or more microalgae, said microalgae being functionalized with Cannabidiol (CBD), wherein said coating promotes cell adhesion, cell proliferation and / or cell differentiation. In a preferred aspect, the microalgae are selected from the phylum of the Heterokonts Heterokontaor Heterokontes. The microalgae can belong to one of the classes of Bacillariophyceae, Chrysophyceae, Phaeophyceae, Oomycetes, Raphidophyceae or Xanthophyceae.

[0031] Among biomaterials, micro- and macroalgae, plants, and natural biominerals and organic polymers can exhibit a unique surface nanotextures, very attractive as next-generation nanomaterials. Furthermore, the structural features of microalgae walls can be easy modified, broadening the plethora of applications.

[0032] Cannabidiol (CBD), a plant-derived cannabinoid without psychoactive activity, appears to be a promising compound with medical applications. Besides its neuroprotective, antiepileptic, anxiolytic, anti-inflammatory, and anticancer properties, CBD can be used in bone tissue cell differentiation for its osteogenic potential.

[0033] In the coating for a metallic dental or orthopedic implants according to the present invention, CBD was used to functionalize the cell surfaces of microalgae. Without being bound to any theory, the functionalization is due to an electrostatic interaction between the microalgae structures and Cannabidiol, allowing a metabolic incorporation.

[0034] In an aspect the invention provides a coating wherein said microalgae are microalgae cells or microalgae extracts. Smooth titanium surfaces, widely used for bone implants, were texturized with CBD-functionalized microalgae cells.

[0035] The effect of cannabidiol, cooperatively with the nanostructured cell walls, on proliferation and osteogenic differentiation of human mesenchymal stem cells was investigated for bone tissue engineering applications. Compared to time consuming and environmentally hazardous procedures previously proposed for bone implants, this innovative one step method has the advantage to nanotexurize smooth titanium surfaces and promote osteogenesis.

[0036] In a further aspect, the coating according to the present invention promotes processes of cell adhesion, cell proliferation and / or cell differentiation which involve one or more cells selected from the group consisting of bone cells, including osteoblasts and mesenchymal stem cells.

[0037] In a further aspect the coating for a metallic dental or orthopedic implants can further comprise a bioactive substance such as a growth factor, peptide, hormone or substance influencing cell adhesion, cell proliferation, cell differentiation and / or bone mineralization.

[0038] Microalgae offer different chemical and physical structures, resistant to harsh conditions, processings, and further functionalization, in principle useful for producin g new generation biomaterials.

[0039] Plant-derived cannabinoids possess wide range of therapeutic applications, due to antiinflammatory and analgesic properties and have been used for years in medical treatment of several pathologies. The pharmacological properties of cannabinoids rely on the interaction with endocannabinoid receptors located throughout the tissues, including dental tissues. Among cannabinoids, cannabidiol (CBD) is a compound without psychoactive activity derived from Cannabis sativa plant. Research targetin g the possibilities of CBD use for stem cells self-renewal, proliferation and differentiati on has been developed. Only in the last years, the role of CBD in bone regeneration and its potential uses in dentistry has been investigated. Therefore, it would appear to be promising to explore the possibilities for future research on the use of CBD for dental devices.

[0040] In a preferred aspect, the coating of the bone implants promotes osteogenesis and the metal of which the implant is made is selected from the group consisting of titanium , zirconium, tantalum or a titanium alloy such as TieAkV (having a composition by weight of approximately V 3,5-=-4,5%, Al 5,5-6,75%, Ti balance to 100%).

[0041] In a second aspect, the use of the coating for a metallic implant is herein described for biomedical and dentistry applications.

[0042] In a referred embodiment, the metallic implant is for a dentistry application, and is selected from the group consisting of a post, a screw, a dental fixture...

[0043] In a third embodiment, the invention relates to a metallic implant having an outer layer, said outer layer being the coating comprising one or more microalgae functionalized with CBD. Preferably said implant consists of titanium, zirconium, or a titanium alloy.

[0044] In one aspect the metallic implant is a dental implant and is preferably selected from the group consisting of a post, a screw, a dental fixture.

[0045] In a further aspect, the invention relates to a process for the preparation of a coated metallic implant, comprising the steps of: a. preparing a culture of living microalgae; b. adding Cannabidiol to the culture of living microalgae of step a. to obtain a mixture; c. allowing the Cannabidiol of the mixture of step b. to be adsorbed by the living microalgae, to obtain a solution of microalgae functionalized with Cannabidiol; d. immersing a metallic implant in the culture of microalgae functionalized with Cannabidiol of step c.; e. obtaining a coated metallic implant.

[0046] In a preferred aspect of the process, step a. of preparing a culture of living microalgae is carried out via a gentle soft cleaning process.

[0047] Preferably, the Cannabidiol which is added to the culture of living microalgae of step b.is in a range from 0.01 to 10 mg / mL, preferably from 0.01 to 5 mg / mL, more preferably from 0.01 to 0.1 mg / mL

[0048] In one aspect steps c. and d. take place in alternation several times, preferably the coating is built up in layers.

[0049] A further aspect of the present invention relates to a coated metallic implant obtainable by the process as herein described.

[0050] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0051] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention.

[0052] Example 1.: In vivo CBD functionalization

[0053] Microalgae cells, belonging to the Heterokonts, were in vivo decorated with CBD (0.01 -0.1 mg / mL) via soaking, adsorbing and active uptake processes. This process was performed in a bioreactor at 18°C (±2°C), 64% relative humidity. The functionalization was performed in green conditions, using enriched sea water under dark:light cycle (12:12 hs) as promoting systems for the uptake.

[0054] Example 2.: Production of titanium microalgae scaffolds

[0055] Titanium (Ti) sheets were activated in a 1 M KOH solution for 20 minutes on a hot plate and rinsed twice with ddH2O, followed by ethanol washing step. Air-dried Ti sheets were then immersed in a solution consisting of anhydrous hexane and diisopropylethylenamine (DIPEA) for few minutes at room temperature. Microalgae decorated with CBD, prepared as described in Example 1 , were seeded on Ti to produce Titanium microalgae scaffolds.

[0056] Results:

[0057] Growth kinetics of microalgae was not hugely affected by the soaking, adsorption and uptake of CBD. The number of microalgae cells remained almost over 60% of the total control cells, reaching also 95% in some samples. The growth rate of CBD-supplemented microalgae appeared slower than untreated microalgae only during the first days. These results confirmed the capability of living microalgae to grow even if the medium is supplemented with CBD. Furthermore, the CBD additions resulted biocompatible with cell growth, avoiding cell death and degradation (Figure 1).

[0058] The CBD uptake from the growth medium was confirmed by GC-MS analysis (Figure 2). In the case of 0.01 mg / mL addition of CBD, the percentage of uptake was not calculable probably due to detection limit. The uptake ranged 68-72% when 0.05 mg / mL and 0.1 mg / mL of CBD were added in the diatom’s growth media.

[0059] Spectroscopic FTIR-ATR investigation confirmed the CBD functionalization of microalgae (Figure3). All the samples exposed to 0.01 mg / mL, 0.05 mg / mLand 0.1 mg / mLof CBD exhibit the characteristic bands of CBD. (3520 cm-1and 3441 cm-1corresponding to the stretching vibration of hydroxyl groups, peak at 3073 cm-1assigned to the C-H stretching vibration of the benzene ring, at 2964 cm -1 , 2924 cm- 1 , 2854 cm-1, and 2828 cm-1denoted for the stretching vibration of methyl and methylene groups at 1623 cm-1, 1581 cm-1, 1513 cm-1, and 1443 cm-1 corresponding to benzene skeleton vibration, at 1375 cm-1denoted for the bending vibration of methyl groups, and at 1214 cm-1for the C-0 stretching vibration).

[0060] CBD-functionalized microalgae were grown on Ti scaffolds in order to improve the roughness of the smooth surface and the organic matter was removed gently from textured surfaces, leaving decorated cell walls over titanium surfaces.

[0061] Bidimensional fluorescence analysis (Figure 4) confirmed that the roughness of Ti was increased due to microalgae grown on Ti surfaces. The biohybrid nanostructured material made of Ti@CBD could be exploited as a scaffold for biomedical applications to enhance cellular adhesivity, promote proliferation of osteoblasts and trigger mesenchymal stem cell differentiation. CBD enhances microalgae, macroalgae, and natural extracts (biominerals, organic polymers) adhesion and stability on active surfaces usually exploited for bone tissue regeneration.

[0062] Furthermore, SEM analysis (Figure 5) of Ti@CBD scaffolds also allowed to recognize the presence of cell walls over surfaces. The presence of microalgae cells on Ti scaffolds could represent a biocompatible approach to improve long-term fixation of Ti implants enhancing osseointegration response in living bone tissue. Easy and affordable design and fabrication of such Ti@CBD scaffolds, for the large availability and large-scale growth of microalgae, can be massively exploited to produce new healthcare devices.

[0063] Example 3.: Cell culture

[0064] Human stem cells (hMS cells commercial line) were cultured in DMEM supplemented with 10% (v / v) FBS, L-glutamine and antibiotics at 37°C and 5% CO2 using polystyrene activated flasks for adherent cells. All the samples for the biological assays were sterilized by soaking them in ethanol and treated under UV. Before each time (7, 14, 21 days), hMSCs were seeded onto the scaffolds starting from a density of 1.6 x 104cells / sample in multi-well plates.

[0065] Example 4.: Real time RT-PCT assay

[0066] Total RNA was isolated from hMSCs after 7-14-21 days and was retrotranscribed . PrimePCR SYBR Green Assay kits (Bio-Rad) of RUNX2 (qHsaCED0044067), SP7 (qHsaCED0003759) and COL1A1 (qHsaCED0043248) were used for the relative gene expression quantification carried out by Real-Time Quantitative PCR. The housekeeping gene glyceraldehydes-3-phosphate dehydrogenase (GAPDH, qHsaCED0038674) was used as an endogenous control. Results:

[0067] The mRN A expression of the three candidate markers (RUNX2, SP7, COL1A1 ) for osteoblast differentiation was examined to evaluate the effects of Ti@CBD on hMSCs differentiation, using quantitative Real Time RT-PCR assay at day 7, 14 and 21. The fold change in the expression of RUNX2, SP7, COL1A1 confirmed the activation of the osteogenic pathway.

Claims

CLAIMS1 . Coating for a metallic dental or orthopedic implant, said coating comprising one or more microalgae, said microalgae being functionalized with Cannabidiol (CBD), wherein said coating promotes cell adhesion, cell proliferation and / or cell differentiation.

2. The coating according to claim 1 , wherein said microalgae are microalgae cells of the phylum of the Heterokonts.

3. The coating according to any one of claims 1 or 2, wherein said processes of cell adhesion, cell proliferation and / orcell differentiation involve one or more cells selected from the group consisting of bone cells, including osteoblasts, and mesenchymal stem cells.

4. The coating according to any one of claims 1 to 3, wherein said coating promotes osteogenesis.

5. The coating according to any one of claims 1 to 4, wherein said metal is selected from the group consisting of titanium, zirconium, tantalum, or of a titanium alloy.

6. The coating according to any one of claims 1 to 5, further comprising a bioactive substance such as a growth factor, peptide, hormone or substance influencing cell adhesion, cell proliferation, cell differentiation and / or bone mineralization.

7. Use of the coating for a metallic implant according to any one of claims 1 to 6, for biomedical and dentistry applications.

8. The use according to claim 7, wherein said metallic implant is for a dentistry application, and is selected from the group consisting of a post, a screw or a dental fixtu re.

9. A metallic implant having an outer layer, said outer layer being the coating according to any one of claims 1 to 6.

10. The metallic implant according to claim 9, said implant consisting of titanium, zirconium, tantalum or a titanium alloy such as TieAkV (having a composition by weight of approximately V 3,5-?4,5%, Al 5,5-?6,75%, Ti balance to 100%).1 1. The metallic implant according to any one of claims 9 to 10, wherein said implant is a dental implant.

12. The metallic implant according to any one of claims 9 to 1 1 , wherein said implant is a dental implant selected from the group consisting of a post, a screw or a dental fixtu re.

13. Process for the preparation of a coated metallic implant, comprising the steps of: a. preparing a culture of living microalgae; b. adding Cannabidiol to the culture of living microalgae of step a. to obtain a mixture; c. allowing the Cannabidiol of the mixture of step b. to be adsorbed by the microalgae; to obtain a culture of microalgae cells functionalized with Cannabidiol; d. immersing a metallic implant culture of microalgae cells functionalized with Cannabidiol of step c.; e. obtaining a coated metallic implant.

14. The process for the preparation of a coated metallic implant according to claim 13, wherein step a. of preparing a culture of living microalgae is carried out via a gentle soft cleaning process.

15. The process for the preparation of a coated metallic implant according to any one of claims 13 or 14, wherein the Cannabidiol added to the culture of living microalgae of step b.is in a range from 0.01 to 0.1 mg / mL.

16. The process according to any one of claims 13 to 15, wherein steps c. and d. take place in alternation several times.

17. The process according to any one of claims 13 to 16, wherein the coating is built up in layers.

18. A coated metallic implant obtainable by the process according to any one of claims 13 to 17.

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