Natural Antibiotic Oregano in Hydroxyapatite-Coated Titanium Reduces Osteoclastic Bone Resorption for Orthopedic and Dental Applications (2024)

2.1. HA Disc Sample Preparation.

Hydroxyapatite (HA) powder was prepared via ethanol mixing (2:3 w/v ratio) with zirconia balls (3:1 ball-to-powder ratio) for 12 h at 80 rpm. Ethanol was left to fully evaporate at 70 °C. The powder was sieved and pressed into discs of 12 mm diameter and 2 mm thickness, utilizing a uniaxial press at 165 MPa for 2 min. Substrate discs were sintered at 1250 °C for 2 h in a muffle furnace. HA discs will be further referred to as HA-D.

2.2. Plasma-Sprayed HA-Coated Ti6Al4V Sample Preparation.

Commercial HA powder, with particle sizes between 175 and 212 μm, was loaded into an axial powder system within a 30 kW inductively coupled radio-frequency plasma spray system (Tekna Plasma System, Canada). Ti6Al4V (Ti64) samples were waterjet-cut into circular samples of 12.5 mm diameter with 2 mm thickness. Prior to plasma coating, Ti64 samples were sandblasted and ultrasonically washed with deionized water followed by ethanol. Plasma operating parameters include 25 kW plate power, 110 mm spray distance from a supersonic plasma nozzle, an argon central gas flow rate of 25 standard 1 min⁻¹ (s.l.p.m.), an argon carrier gas flow rate of 13 s.l.p.m., a sheath gas of argon 60 s.l.p.m. and hydrogen 6 s.l.p.m., and a chamber pressure of 5 lb-force per square inch gage (p.s.i.g). HA plasma samples will be further referred to as HA-P. The coating thickness is maintained with an average between 60 and 80 μm.

2.3. Carvacrol and Thymol Extraction from Oregano.

Dried oregano leaves (McCormick) were crushed using a mortar and pestle. The powdered leaves were immersed in 95% ethanol (5% deionized water) at a concentration of 20 mg/mL and vortexed for 30 s. The solution was filtered through Whatman No. 1 filter paper through a Buchner funnel to remove any undissolved leaf remnants and stems (Figure S1). The extraction was analyzed using UV–vis spectroscopy via a Biotek Synergy 2 SLFPTAD microplate reader (Biotek Winooski, VT) to assess the compounds derived. A ranged wavelength scan was performed between 250–300 and 500–700 nm. The extraction was also analyzed using proton ¹H NMR spectroscopy. Deuterated water was supplemented into the extraction prior to use in a Bruker DRX 400 MHz spectrometer.

2.5. Effects of Carvacrol and Thymol on S. epidermidis Disc Diffusion Test.

Antibacterial properties of carvacrol and thymol were assessed using a disc diffusion test with S. epidermidis (Carolina Biological Supply Company, Burlington, NC). Various concentrations of oregano extract (10, 200, 500, 800, 1000 μg/mL) were deposited onto antibiotic sensitivity discs. The bacteria were purchased as a MicroKwik Culture pathogen vial and rehydrated per company specifications. After 2 d incubation at 37 °C, S. epidermidis was inoculated onto nutrient agar plates with discs. Plates were imaged and zone of inhibition was analyzed after incubation for 24 h at 37 °C.

2.6. Effects of Carvacrol and Thymol In Vitro on Human Fetal Osteoblast Cells.

HA-D samples were sterilized in an autoclave at 121 °C for 1 h, loaded with carvacrol/thymol (1000 μg) in a sterilized environment, and left to dry. Human fetal osteoblast cells (hFOB 1.19, ATCC, Manassas, VA) were seeded onto the drug-loaded discs at a density of 25 000 cells per sample and submersed in a cell media of Ham’s F12 and Dulbecco’s modified Eagle’s medium (DMEM/F12, Sigma-Aldrich, St. Louis, MO) mixed with 1.2 mg/mL of sodium bicarbonate and 0.3 mg/mL G418 (Sigma-Aldrich, St. Louis, MO) in DI water and supplemented with 10% fetal bovine serum (FBS, ATCC, Manassas, VA) as well as 0.1% penicillin–streptomycin. Cultures were incubated at 34 °C in an atmosphere of 5% CO₂. The media was changed every 2–3 days throughout the entire experiment. To characterize the osteoblast cell viability, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was employed with time points of 3, 7, and 11 days. The samples were immersed in a solution of 100 μL of MTT (Sigma-Aldrich, St. Louis, MO) solution and 900 μL of cell media for 2 h at 34 °C. This solution was removed and replaced with 600 μL of solubilizer solution composed of 10% Triton X-100, 0.1 N HCl, and isopropanol to dissolve the formazan crystals formed from the reduction of MTT through the cellular enzyme reaction. Each sample solution was assessed using a Biotek Synergy 2 SLFPTAD microplate reader (Biotek, Winooski, VT) with 100 μL of the solution at 570 nm. Scanning electron microscopy (SEM) images were taken to show the cellular morphology of the culture. The samples were first fixed in a solution composed of 2% paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer and incubated at 4 °C. Following preliminary fixation, the samples were rinsed with 0.1 M phosphate buffer and fixed with 2% osmium tetroxide for 2 h. The samples were then rinsed with 0.1 M phosphate buffer, deionized water (3×), and a series of ethanol dehydration of 30, 50, 70, 90, and 100% (3×) with a final dehydration using hexamethyldisilane (2×). The samples were gold sputter-coated prior to imaging with SEM.

The following hFOB culture was performed using identical methods; however, HA-P samples with a loading of 500 μg of carvacrol/thymol were utilized due to the preceding results. A higher cell density of 40 000 cells per sample was used for HA-P samples due to the higher possibility of cellular wash-off with HA-coated samples compared to that of pressed disc samples.

2.7. Effects of Carvacrol and Thymol In Vitro on Osteoclast Cells (THP1 Monocytes).

Osteoclast cells were differentiated from THP1 monocytes (ATCC, Manassas, VA). Growth media and plating were performed via the manufacturer’s specifications. Monocytes were seeded onto HA-D samples loaded with 500 μg of carvacrol/thymol at a density of 10 000 cells per sample. Once seeded onto control and drug-loaded samples, differentiation media was used through the remainder of the study. Differentiation media was composed of 40 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO) and 10 ng/mL receptor activator of nuclear factor kappa-B ligand (RANKL) with RPMI-1640 and FBS. Cultures were kept at 37 °C and 5% CO₂, and differentiation media was changed every 3 days. Characterizations at time points of 7 and 16 days include MTT assay for cell viability quantification, telomerase-repeated amplification protocol (TRAP) assay (Cayman Chemical, Ann Arbor, MI), and SEM imaging of cell morphology as well as resorption pit formation.

2.8. Statistical Analysis.

Quantitative characterizations are presented as mean ± standard deviation. Measurements were taken in triplicate with a p value <0.05 considered significant and indicated with an asterisk within figures. The statistical tests were performed with one-way ANOVA and post hoc Tukey–Krammer analyses.

3.1. Characterization of Oregano Extraction.

For the UV–vis analysis, a scan from 200 to 900 nm is performed with two strong peaks identified at 276 (carvacrol/thymol) and 665 nm (carvacrol) with a weak peak at 533 nm (thymol) (Figure S1). Literature references specifically for oregano extraction and its phenolic compounds are limited; therefore, there is a lack of available standards. However, phenolic-type compounds can be characterized by the absorption maxima between 260 and 330 nm.²³ Pure carvacrol has two identifiable peaks around 275–278 and 653 nm.^24–26 Pure thymol has two identifiable peaks around 274–277 and 531 nm.^27–29 In this study, all oregano extraction solution is analyzed at 665 nm due to this wavelength showcasing the highest intensity among the three peaks identified. A small shift in peak values compared to the literature can naturally occur from differing extraction processes, purity, source of oregano leaves, and well-plate effects on wavelength readings. There is the potential for compound interaction, as this extraction is not purified like a laboratory standard carvacrol or thymol. However, ¹H NMR spectroscopy did identify and further support the successful extraction of these active terpenoid compounds. The hydrogens of both carvacrol and thymol isomers as well as other characteristic peaks are revealed, corroborating carvacrol and thymol as active compounds extracted from the dried oregano leaves^30–33 (Figure 1).

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Figure 1.

¹H NMR spectra of oregano extract. Identification of carvacrol and thymol isomers.^30–33

3.2. Carvacrol and Thymol In Vitro Release Study.

To assess the coating surface morphology and degradation characteristics post carvacrol/thymol loading, SEM images were taken of an HA plasma control sample and a 500 μg oregano-HA-P sample (Figure 2). It is imperative to assess degradation characteristics of the HA coating post drug loading due to the inherent amorphous phases that can occur from plasma spray coating fabrication. The high temperatures of the plasma spray can result in rapid cooling of the coating, leading to less crystalline coatings. These amorphous phases can increase the dissolution rate of coatings, thereby leading to a reduction in stability, especially in vivo.^19,21,34 Drug loading should not further add to the coating dissolution properties and therefore should not cause any degradation to the coating. As seen in the SEM micrographs, no changes are visible regarding surface morphology and no degradation is observed post carvacrol/thymol loading indicating feasibility and stability of the drug-loaded system.

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Figure 2.

SEM micrographs of the carvacrol/thymol extraction from oregano loaded onto plasma spray HA-coated Ti6Al4V substrates. (a)HA plasma (HA-P) control samples. (b) Carvacrol/thymol-loaded HA plasma sample showing no changes in coating surface morphology nor signs of any degradation.

An initial release kinetic optimization study is performed with carvacrol/thymol loaded into HA-D samples to determine if the hydrophobic drug compound could be successfully released in the aqueous buffer solutions with and without polymer addition. The metabolic and physiological environment of the body already places a feasibility hurdle for natural compounds because it typically hinders the practical application and limits the bioavailability of natural medicinal compounds.^35,36 Aiding in the drug release capability and bioavailability of the compound can be facilitated with the use of other biomaterials like polymers. In this study from HA-D, carvacrol/thymol release is successful with all compositions including just oregano extraction (no polymer), with a PCL coating, and with PCL/PEG in pH 7.4 (Figure 3a). As expected, due to the enhanced hydrophilicity aided by PEG, the release of carvacrol/thymol from PCL/PEG-loaded samples reached full release within 10 days. This is much earlier than without polymer and with PCL. Also as expected, with a PCL coating, burst release is reduced significantly in early time points and the overall release of the drug is lower.

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Figure 3.

Carvacrol/thymol release in pH 7.4 and 5 from HA discs (a, b) and HA plasma-coated (c, d) samples. (a) In pH 7.4, the release from HA discs is 100% with PCL/PEG in 10 days, 95% without polymer in 50 days, and 60% with a PCL coating in 50 days. Fitted to a first-order release kinetics. (b) In pH 5, a shift to zero-order release is 75% at 12 h. (c) Full release of carvacrol/thymol is achieved in pH 7.4 from HA plasma-coated samples in 3 days and is fitted for a first-order kinetics. (d) Full release is again achieved in pH 5 from HA plasma-coated samples in 3 days and is fitted for a first-order kinetics. In pH 7.4, a faster release is seen from HA-P, but in pH 5, a faster release is seen from HA-D, with a differing mechanism. In addition to pH change, the shift is caused by differing process parameters leaving HA-D dense and HA-P porous, altering HA matrix surface area properties. In pH 5, the release from disc samples is determined to be mostly diffusion-based, whereas release from plasma samples is assessed to be dissolution/degradation-based, a supportive observation for a difference in release kinetics model. Successful release from the HA plasma coating indicates clinical relevance and ability to be used in an orthopedic or dental implant.

These findings are in line with previous works, with one focused on modulating the release of curcumin, a hydrophobic medicinal compound extracted from turmeric root.^37,38 Once carvacrol/thymol release is identified as successful without polymer despite the low water solubility, the remaining release experiments are performed without polymer. This is to reduce the complexity of the system and aid in accurately assessing the effects of carvacrol/thymol in vitro on osteoblast and osteoclast cells, without interference of other compounds interacting.

All release from HA-D (Figure 3a,​,b)b) and subsequently HA-P (Figure 3c,​,d)d) is fitted to either a zero-order or a first-order release kinetic model.³⁹ Although not perfect fits to the models, the trend aids in deducing the base release mechanism of carvacrol/thymol from each respective sample in each buffer solution. For HA-D in pH 7.4, being a close fit for the first-order release across 50 d (1200 h) indicates a diffusion-based release mechanism because the disc does not visibly degrade in the buffer medium due to the lack of/limited sample porosity. However, for HA-D in pH 5, a zero-order release showcases a constant release of carvacrol/thymol, which occurs much earlier and quicker comparatively to pH 7.4. This is expected due to the acidic environment effect on calcium phosphates and in drug delivery. Although a different system, another study also showed a higher release of carvacrol/thymol in acetate buffer compared to that of phosphate and a basic buffer medium.⁴⁰

The shift in the carvacrol/thymol release mechanism, from first order in pH 7.4 to zero order in pH 5, is not seen in HA plasma-coated Ti6Al4V metal samples but is seen in HA disc samples. For HA-P release, both buffer solutions fit to first-order release models, but in analyzing the SEM micrographs post release (Figure 4), it is evident that the release in pH 7.4 is still dominated by a diffusion-based mechanism, whereas in pH 5, the release is dominated by a matrix dissolution/degradation-based mechanism. Particles are much more easily viewed on phosphate buffer samples compared to those on acetate buffer and it is noted that carvacrol/thymol helped reduce natural degradation of a pure HA-coated sample in pH 5. The release may appear to be faster from HA-P samples compared to that from HA-D samples in pH 7.4, but upon closer analysis, for both 1000 μg oregano-HA-D and 500 μg oregano-HA-P, approximately 500 μg is released by 3 days. In both cases, the mechanism is identified as diffusion-based first order but a shift from zero to first order is seen in pH 5 between HA-D and HA-P. The assessment of release from disc samples to be mostly diffusion-based, whereas release from plasma samples is dissolution/degradation-based, additionally supports the difference in release kinetics model. This shift can occur because the acidic buffer increases the release of carvacrol/thymol compared to pH 7.4, in combination with the differing processing parameters.⁴⁰ Processing differences between discs and plasma coatings will result in differing porosity because pressed discs are sintered, whereas plasma-coated samples retain a variable powder surface morphology. The denser HA disc matrix shows zero order due to the lower surface energy of the hydrophobic surface as compared to the porous HA coating, which shows first-order release. Porosity will increase surface area, wettability, and surface energy, ultimately altering how carvacrol/thymol interacts within the hydroxyapatite system matrix. Surface area has been shown to play a role in affecting drug release kinetics.^41,42 Additionally, the majority of drugs release in first-order kinetics, which depends on the concentration and the rate is proportional to the amount of drug released.⁴³

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Figure 4.

HA plasma and carvacrol/thymol-loaded HA plasma samples after the release kinetic study in phosphate-buffered saline (pH 7.4) and acetate buffer (pH 5). (a,b) HA-P and carvacrol/thymol-loaded HA-P samples used in pH 7.4, respectively. (c,d) HA-P and carvacrol/thymol loaded HA-P samples used in pH 5, respectively. The images show comparable degradation in pH 7.4 between carvacrol/thymol treatment to control HA but a reduction in degradation with carvacrol/thymol-loaded sample compared to that of HA control in pH 5. The microstructure of control HA in pH 5 is more degraded, as expected, compared to that of control HA in pH 7.4. This degree of degradation is less severe when comparing carvacrol/thymol treatment in pH 5–7.4.

3.3. Effects of Carvacrol and Thymol on S. epidermidis Disc Diffusion Test.

Carvacrol and thymol can inhibit the growth of S. epidermidis bacteria as seen by the ~40 mm² area zone of inhibition made from the disc diffusion test (Figure 5). A control disc or a disc that is incapable of producing a zone of inhibition would simply have a bacterial colony around the disc with no clear zone.¹⁶ Carvacrol is well known as a natural antibiotic and has been shown to reduce the growth of E. coli, Pseudomonas aeruginosa, Salmonella pullorum, and Staphylococcus aureus, as well as many others.^9–11 One work has suggested that the mechanism of terpenoids on bacterial membranes, leading to growth inhibition, is stimulated by the lipophilic properties of the terpenes as well as the functional groups with their associated aqueous solubility.^44,45 At the phospholipid bilayer of the bacterial cell is the site of the mechanistic action of carvacrol that can affect or inhibit electron transport, protein translocation, phosphorylation, and other enzymatic reactions.⁴⁵

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Figure 5.

See Also

9 Benefits and Uses of Oregano Oil

Disc diffusion test. Oregano extract of carvacrol and thymol is able to inhibit the growth of S. epidermidis bacteria as showcased by the disc diffusion test where a visible zone of inhibition can be seen. All other compositions were successful; however, 1000 μg/mL was the most prominent.

3.4. Effects of Carvacrol and Thymol In Vitro on Osteoblast and Osteoclast Cells.

Little is known on the effects of carvacrol and thymol on osteoblast and osteoclast cells, and the following cultures provide supporting evidence that oregano extractants can positively affect bone healing. Carvacrol/thymol is nontoxic with no effect on proliferation toward osteoblast cells; however, it does influence the reduction of osteoclast activity further, inhibiting resorption. Results of the 1000 μg oregano-HA-D hFOB culture reveal a reduction in proliferation, however, not beyond the cytotoxicity limit, as well as healthy cellular morphology is seen in both control and treatment at all time points (Figure 6). Cytotoxicity is defined as a threshold of 70% viability in the first 24 h.⁴⁶ With the 500 μg oregano-HA-P hFOB culture, a reduction in MTT optical density is not seen with the reduced carvacrol/thymol loading and continued healthy cellular morphology is observed in all samples at all time points (Figure 7).

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Figure 6.

Thousand micrograms of oregano-HA-D hFOB culture. (a) MTT quantification assay. (b–d) Days 3, 7, and 11 SEM micrographs of HA-D and carvacrol/thymol-loaded HA-D, respectively. Although not cytotoxic by significant difference, the viability of the hFOB cells is hindered by the high concentration of carvacrol/thymol. Healthy cell morphology is seen in all samples.

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Figure 7.

Five hundred micrograms of oregano-HA-P hFOB culture. (a) MTT quantification assay. (b–d) Days 3, 7, and 11 SEM micrographs of HA-P and carvacrol/thymol-loaded HA-P, respectively. Cytotoxicity is not seen, and healthy hFOB cell morphology is observed in all samples.

The osteoclast culture reveals a decrease in optical density at day 7 with carvacrol/thymol; however, it reveals an increase by day 16 (Figure 8). Although initially deceptive on identifying a trend, the SEM micrographs of the culture reveal more of the story of what occurred during the culture. At day 7, healthy osteoclast and preosteoclast cells can be seen all over control HA-D samples, resembling those seen in previous osteoclast studies,⁴⁷ whereas on carvacrol/thymol-loaded samples, a large cell clump with no cellular extensions is observed with no other cellular morphology found on the sample surface. This indicates that the osteoclasts did not attach to the carvacrol/thymol-loaded HA surface well. The cells did not migrate or spread and perhaps became necrosed in that one location. This explains the reduced optical density compared to control because the number of attached, viable cells is significantly affected. From the release analysis in pH 7.4, more than half of the loaded carvacrol/thymol is released at 7 days, at approximately 80%. This illustrates a negative correlation between carvacrol/thymol release and osteoclast attachment ability.

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Figure 8.

Osteoclast cell viability and morphology. (a) MTT quantification assay showing that the optical density is lower for carvacrol/thymol-loaded samples at day 7 but an increase at day 16, which is further explained by the SEM micrographs. (b) Day 7 SEM micrographs show an inability for osteoclast cells to attach properly to the carvacrol/thymol treatment surface, as seen by the large cell clump with no cellular extensions. No other cell morphologies are seen on the 500 μg oregano-HA-D sample surface. On control HA-D, round, healthy preosteoclast cells can be seen all over the sample surface. (c) Day 16 SEM micrographs show resorption pits all over the HA-D surface, indicating a healthy apoptosis cycle where osteoclast cells have successfully resorbed the calcium phosphate surface and are removed during the culture. Large osteoclast morphologies are also seen on control HA-D. Due to the inhibited proliferation and extension on carvacrol/thymol samples, the optical density is higher because necrosis has occurred, limiting the ability for cells to resorb the calcium phosphate surface, reach normal apoptosis, and be removed during culture. A comparatively poorer osteoclast cell with detached edges can be seen in the SEM micrograph (n = 3, p < 0.05, *).

Only 10% more release for a total 90% release is seen by day 16, indicating the osteoclast inhibition from carvacrol/thymol is significantly reduced. This explains the increase in MTT optical density due to less available drug. In addition, the SEM images of control HA-D samples show resorption pits all over the sample surface, which indicates a healthy apoptosis cycle of osteoclast cells where the cells had attached, successfully resorbed calcium phosphate, and detached from the surface. There are also large osteoclast morphologies seen that had attached and spread across the surface. This is in stark contrast with carvacrol/thymol-loaded samples as no resorption pits were visible and much smaller, poorly attached cell morphologies could be found on the sample surface. The reason for the additional optical density value in the MTT assay could be attributed to necrosed cell bodies which did not properly detach.

This theory for the increase in MTT between days 7 and 16 with carvacrol/thymol compared to that of control is further corroborated by the significantly reduced TRAP expression by day 16 induced by carvacrol/thymol (Figure 9). TRAP, an enzyme known for roles in bone and the immune system, is historically used as a histochemical marker for osteoclasts.^48,49 Osteoclasts secrete TRAP during bone resorption, and this marker correlates with resorption behavior.⁵⁰ A significant reduction in TRAP activity indicates that the osteoclast cells have become distinctly less viable and/or are not properly attached to the surface; therefore, they are unable to resorb the HA and release TRAP. The quantified assays together also support this reduced cell resorption potential, where TRAP/Cell is reduced about 15% with oregano treatment. This is captured by the significantly smaller-in-diameter and depth severity resorption pits found on carvacrol/thymol-loaded samples as compared to those on control. Control sample resorption pits are approximately 50–75 μm in diameter, whereas carvacrol/thymol resorption pits are approximately 10–35 μm in diameter. One final indicator of poor attachment of osteoclast cells to carvacrol/thymol-loaded samples is the number of dead cells and cell particulates found at the bottom of the wells that held the samples during culture (Figure S2). Control HA samples show hardly any dead cell debris in the well, whereas carvacrol/thymol samples left behind many dead cells and cell particulates. This finding corroborates the SEM micrographs regarding carvacrol/thymol-affected osteoclast morphology where cells are in fewer frequency and poorer attachment compared to control. It has been reported that a reduction in osteoclast activity induced by carvacrol/thymol occurs through suppression of RANKL.¹⁴

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Figure 9.

Osteoclast TRAP assay and resorption pit formation. (a) TRAP activity is near-identical between control and carvacrol/thymol-loaded samples in day 7. (b) In contrast, day 16 TRAP activity has significantly been reduced through carvacrol/thymol. This is corroborated by the cell morphologies seen prior due to the cells being unable to attach properly to the sample surface. Without proper attachment, TRAP cannot be produced because the osteoclast cells have become less viable and are unable to resorb the HA surface. (c) Resorption pit SEM micrographs show control HA-D samples with larger, more frequent resorption pits at approximately 50–75 μm in diameter, whereas carvacrol/thymol treatment shows reduced pit formation at approximately 10–35 μm in diameter (n = 3, p < 0.05, *).

Supplementary information

The authors declare no competing financial interest.

ASSOCIATED CONTENT

Supporting Information

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.0c14993.

Extraction of carvacrol and thymol with UV–vis analysis (Figure S1);

and microscope images of osteoclast cell culture wells (Figure S2) (PDF)

Complete contact information is available at: https://pubs.acs.org/10.1021/acsami.0c14993

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