Synthesis and characterizations of hydrogel + T8IC + laser + BMP-2
T8IC is a novel natural semiconducting materials, primarily utilized in excessive efficiency natural photovoltaic machine. T8IC nano-particles (T8IC NPs) had been synthesized by way of reprecipitation. The Zeta potential of T8IC NPs was 21.9 ± 1.12 mV. It was homogeneously distributed and saved steady in PBS and DMEM for 14 days (Determine S1A). The absorption spectrum of T8IC NPs was 600 ~ 900 nm (Determine S1B). It belonged to the second near-infrared area spectrum (NIR-II, 900 ~ 1700 nm), of which the emission fluorescence spectrum was 900 ~ 1100 nm (Determine S1C). The TEM picture exhibited spherical morphology of T8IC NPs (Determine S1D). The common diameter of T8IC NPs was about 160 nm (Determine S1E-S1F). Apart from, T8IC NPs may produce ROS after 808 nm laser irradiation, suggesting an excellent potential for PDT [48]. Each PTT and PDT of T8IC NPs exhibited good tumor elimination when mixed with sorafenib in our earlier research [48].
The translucent chitosan/β-glutamine/gelatin pre-gel was injectable and flowed like a viscous liquid. Apparently, the sol-gel transition occurred when the temperature was round 45 ℃ inside 2 minutes (Fig. 1A-1B). Scanning electron microscope (SEM) photos revealed porous construction inside the freeze-dried hydrogel (Fig. 1C), which facilitated the storage and launch of medication and cell migration [15]. The rheological properties of Hydrogel + T8IC + Laser + BMP-2 was investigated by way of rheological assessments. The pressure dependent oscillatory rheology experiments (Fig. 1D) revealed that the storage modulus (G’) and the loss modulus (G’’) remained fixed when pressure was beneath 100% whereas the frequency was 1 Hz. Moreover, the G’ was over 100 Pa and far greater than the G’’, indicating steady hydrogel networks fashioned. As well as, the viscosity of hydrogel decreased sharply with rising shear fee (Fig. 1E), implying a shear thinning property, which is helpful for additional injection [50]. The temperature dependent oscillatory rheology experiment (Fig. 1F) was additionally performed and when the temperature was beneath 44.69 ℃, the worth of G’ and G’’ was steady and G’’ was greater than that of G’. Whereas modulus of G’ and G’’ elevated with the temperature sharply and the G’ and G’’ crossed at a temperature of 44.69 ℃. Then, the worth of G’ was greater than that of G’’, implying the sol-gel transition. Thus, such thermosensitive hydrogel is simpler to be injected into bone defects with irregular shapes because of the periodontitis. The load loss (%) of Hydrogel + T8IC + Laser + BMP-2 elevated with time at room temperature (24 ℃) and 37 ℃ (Fig. 1G). 82% weight reduction at room temperature (24 ℃) after 10 days and 93% weight reduction at 37 ℃ after 14 days. So as to consider the efficient laser (808 nm) depth penetration of the hydrogel, the residual laser depth was detected together with the hydrogel depth (Fig. 1H). The laser depth decreased with the hydrogel depth. 62% residual laser depth was detected when the hydrogel depth was 20 mm, indicating that hydrogel mixed with T8IC NPs was succesful for the remedy of periodontitis with deep periodontal pocket.
The hydrogel has been broadly used because the scaffold and drug service for managed drug launch in craniofacial tissue engineering, contributing to delivering antibiotics, development components, chemotherapy medication, vaccines and anti inflammatory brokers to intention tissues and cells [51,52,53]. Twin-functional supply methods current with synergistic results together with inhibiting bacterial development, facilitating wound therapeutic, reinforcing bone regeneration and decreasing inflammatory responses. Within the current research, osteo-inductive agent (BMP-2) and NIR-II phototherapy brokers (T8IC NPs) had been utilized inside the hydrogel. The BMP-2 releasing from the hydrogel (Fig. 1I) had been monitored. Cumulative BMP-2 launch of Hydrogel + T8IC + Laser + BMP-2 was greater than that of Hydrogel + T8IC + BMP-2.
A superb photothermal conversion skill of Hydrogel + T8IC + Laser + BMP-2 was detected below totally different concentrations of T8IC NPs and totally different laser energy (808 nm) (Fig. 1J Ok). The photothermal impact was focus and energy dependent. It was additionally offered with an excellent photothermal stability (Fig. 1L).
Synthesis and characterizations of Hydrogel + T8IC + Laser + BMP-2. A-B: Consultant pictures of sol-gel transition. C: Scanning electron microscopy of Hydrogel + T8IC + Laser + BMP-2. D: Variations of storage and loss moduli (G’ and G’’, respectively) versus pressure (%). E: Viscosity versus shear fee (1/s). F: The temperature dependent oscillatory rheology experiment. G: The load loss (%) of Hydrogel + T8IC + Laser + BMP-2. H: Laser (808 nm) depth penetration of Hydrogel + T8IC + Laser + BMP-2. I: Cumulative launch of BMP-2. J: Photothermal results of Hydrogel + T8IC + Laser + BMP-2 with totally different focus of T8IC NPs. Ok: Photothermal results at totally different laser energy. L: Photothermal stability of Hydrogel + T8IC + Laser + BMP-2.
Photothermal results on the cell proliferation in vitro
On this research, the cytotoxicity of T8IC NPs was assessed by way of CCK-8 assay. The cell viability of MC3T3-E1 cells wasn’t influenced by T8IC NPs when the focus was below 50 µg/ml with out irradiation (Fig. 2A), implying an excellent biosafety. As for the photothermal results, Hydrogel + T8IC + Laser + BMP-2 exhibited exceptional cell toxicity with 808 nm laser irradiation (1.5 W/cm2) when the temperature was over 45 ℃. The cell viability decreased to 73%, 45%, 6% after temperature reached to 45 ℃, 50 ℃ and 55 ℃, respectively (Fig. 2B). Photothermal impact of Hydrogel + T8IC + Laser + BMP-2 was additionally monitored in 96-well plates (Fig. 2C). The temperature of Hydrogel + T8IC + Laser + BMP-2 raised to 45 ℃ inside 4 min, whereas it was solely as much as 28.6 ℃ within the Hydrogel + Laser group. The stay/lifeless cell staining additionally proved that the upper temperature was and extra red-fluorescence detected, implying much less cells alive (Fig. 2D). Thus, a relative gentle temperature needs to be chosen to guard cells from the hyperthermia and in addition meet the necessity of bactericidal impact within the subsequent research.
Photothermal results on the proliferation of MC3T3-E1 cells. A: Completely different concentrations of T8IC NPs on the proliferation of MC3T3-E1 cells. B: Photothermal impact on the proliferation of MC3T3-E1 cells, H: hydrogel, T: T8IC, L: Laser, B: BMP-2. C: Photothermal results in 96-well plates. D: The Dwell-dead cell staining of MC3T3-E1 cells (inexperienced: stay cells, purple: lifeless cells), scale bar: 100 μm
Antibacterial impact in vitro
With excessive prevalence of antibiotic-resistant micro organism, many novel nano-material based mostly antibacterial methods has been created on the proper time [53, 54]. Nonetheless, the facet impact of excessive temperature created by PTT on the encircling tissue could restrict its medical software. Thus, it’s significant to hunt a gentle temperature which may kill the bacterial and in addition scale back the harm to the encircling tissue. Many research have elucidated that gentle temperature PTT can obtain unbelievable therapeutic efficiency to allow sensible biomedical functions. Though there are totally different thresholds of gentle temperature PTT, it’s well known that many of the gentle hyperthermia remedies are performed beneath 48 ℃ [55].
Firstly, we assessed the temperature on the bacterial inhibition. The hydrogel and Hydrogel + BMP-2 had no impact on the P. gingivalis proliferation (Fig. 3A). The bacterial quantity decreased at a management group of 37 ℃ after laser irradiation, indicating that PDT performs an necessary position within the bacterial ablation. Apart from, the eradication charges of pathogens elevated with the temperature. The antibacterial exercise of Hydrogel + T8IC + Laser + BMP-2 (45 ℃) was enhanced in contrast with Hydrogel + T8IC + Laser + BMP-2 (37 ℃), however nonetheless not sufficient to fully get rid of biofilms. Thus, we deliberate to advertise the PDT to facilitate the antibacterial impact as an alternative of accelerating the temperature, which was dangerous to the cell proliferation. Often, H2O2 was discovered to be an excellent photosensitizer, which was generally used as periodontal pocket rinse answer after periodontal preliminary remedy. Though H2O2 can successfully kill micro organism by attacking and destroying DNAs and proteins, a excessive focus of H2O2 exhibited poisonous impact to the traditional tissue. After catalyzed into reactive oxygen species (ROS), H2O2 manifested higher antibacterial effectivity [25, 56]. Due to this fact, a decrease focus of H2O2 and enhanced antibacterial effectivity was designed. DBPF probe was used to evaluate modifications of ROS content material in Hydrogel + T8IC + Laser + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 below 808 nm laser irradiation. As proven in Fig. 3B, the ROS yield was calculated by monitoring the oxidation of DPBF at 418 nm in dichloromethane [57]. The extra DBPF loss after including H2O2, implying extra ROS generated and a greater PDT impact. Different research had proven that the mixture of PDT and PTT may destroy the bacterial membranes and improve bacterial eradication [14, 58, 59]. Consequently, the antibacterial impact had been promoted within the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45 ℃) group in comparison with the Hydrogel + T8IC + Laser (45 ℃) group (Fig. 3C), suggesting that synergistic impact of an enhanced PDT and a gentle temperature PTT (45 ℃) may purchase a greater bacterial eradication. There was no vital distinction amongst management, Hydrogel, Hydrogel + H2O2 and Hydrogel + BMP-2 after 10 days tradition by the colony forming unit assay (Fig. 3D). Each Hydrogel + T8IC + Laser (45 ℃) and Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45 ℃) had considerably much less P. gingivalis colonies, whereas the pathogens within the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45 ℃) had been almost fully eradicated. As well as, P. gingivalis suspended inside the BHI medium, so 3D photos of the stay/lifeless (inexperienced/purple) bacterial fluorescence staining had been constructed as much as detect the biofilm viability of P. gingivalis (Fig. 3E). Completely solely inexperienced fluorescence was noticed within the group of management, Hydrogel, Hydrogel + H2O2 and Hydrogel + BMP-2, indicating biofilms had been intact. A small quantity of purple fluorescence was detected in Hydrogel + T8IC + Laser (45 ℃) and the biofilms had been virtually stained purple in Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45 ℃), in keeping with the colony forming unit assay. Normally, the phototherapy generated from Hydrogel + T8IC + Laser + BMP-2 + H2O2 together with enhanced PDT and gentle PTT, exhibited a superior bactericidal impact. Due to this fact, we lastly chosen 45 ℃ as a gentle temperature within the following research.
The antibacterial impact in vitro. A: OD worth of P. gingivalis. at 600 nm. H: hydrogel, T: T8IC, L: Laser, B: BMP-2. B: ROS technology after including H2O2, s: second. C: OD worth of P. gingivalis. at 600 nm below totally different situations, h: hour. D: Images of P. gingivalis colonies after totally different remedies. E: 3D photos of stay/lifeless bacterial staining (inexperienced: stay cells, purple: lifeless cells) after totally different remedies
Osteogenic impact in vitro
So as to detect the impact of enhanced PDT and gentle PTT to the cell proliferation, the cell spreading of MC3T3-E1 cells after totally different remedies was monitored. As proven in Fig. 4A, Hydrogel, Hydrogel + H2O2 and Hydrogel + BMP-2 had much less impact on the actin filaments labelled by phalloidine (purple), whereas cell shrinkage and detachment within the Hydrogel + T8IC + Laser (45 ℃) and Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45℃) had been noticed because of the mixed results of PTT and PDT. Delightfully, the cell regained the bioactivity, cell spreading and attachment after one other 24-hour tradition. Apart from, though the proliferation of MC3T3-E1 cells was inhibited in Hydrogel + T8IC + Laser and Hydrogel + T8IC + Laser + BMP-2 + H2O2 1 day after remedy, the cell viability had resuscitated 3 days after remedy (Fig. 4B).
Though BMP-2 enhances the recruitment and angiogenesis of osteoblast precursor cells and has attracted a lot consideration for its glorious bone-inducing skill [40, 41], its industrial use is proscribed by two main features: firstly, BMP-2 has a brief half-life (solely 7 min) within the physiological atmosphere and a excessive dose of BMP-2 is required to be loaded into these scaffolds to unravel its instability and fast inactivation, which can lead to vital prices and an elevated danger of negative effects, together with irritation, nerve harm, ectopic ossification and tumorigenesis [60,61,62]. Secondly, absorbable collagen sponge and calcium phosphate [63, 64] are at the moment the one two BMP-2 carriers accepted for medical use. Nonetheless, due to the low affinity of those vectors for BMP-2 and their low retention fee, the usage of excessive doses may also exacerbate native and systemic adversarial results. To beat these issues and optimize the bone therapeutic course of, varied approaches have centered on bettering the loading fee of BMP-2 whereas sustaining its organic exercise. Amongst them, injectable hydrogels with 3D networks have turn into the first alternative for BMP-2 carriers in tissue engineering resulting from their excessive water-absorption, injectable skill, encapsulation skill, biocompatibility and biodegradability [42,43,44].
Osteoblast differentiation was assessed by mRNA expression (Determine S2), quantitative evaluation of ALP exercise, ALP staining and alizarin purple staining. After 4 days tradition, gene expression of osteogenic mRNAs (early osteogenic marker: ALP, particular osteogenic differentiation markers within the earlier stage: Runx2, late osteogenic marker: OCN, non-collagenous proteins: OPN, extracellular matrix protein: Col-I) was a lot greater within the Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 (Fig. 4C-G, P < 0.05). Whereas the mRNA expression (ALP, OCN and OPN) of Hydrogel + T8IC + Laser + BMP-2 + H2O2 was considerably greater than that of Hydrogel + BMP-2 (P < 0.05), indicating that PTT may induce the discharge of BMP-2 and promote osteogenic gene expression, as proven in Fig. 1I.
Cytotoxicity and osteogenic impact in vitro. A: The modifications of cytoskeleton after totally different remedies, yellow arrow: cell shrinkage, scale bar: 100 μm. B: Cell viability 1 day and three days after totally different remedies. C-G: mRNA expression of osteogenic marker (ALP, Runx2, OCN, OPN, Col-I).
The ALP staining (Fig. 5A) and alizarin purple staining (Fig. 5B) had been in keeping with the outcomes of PCR. Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 exhibited intensified staining of ALP and mineralized nodules. As well as, the ALP exercise of Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 was a lot greater than different teams after each 4 days and seven days tradition (Fig. 5C, P < 0.05). The ALP exercise of Hydrogel + T8IC + Laser + BMP-2 + H2O2 after 7 days tradition was greater than that of Hydrogel + BMP-2. Moreover, the microscopic pictures of alizarin purple staining confirmed there was highest density of mineralized nodules deposition within the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (Fig. 5D). All these outcomes proved that the gentle PTT and enhanced PDT generated by Hydrogel + T8IC + Laser + BMP-2 + H2O2 could not have an effect on the discharge and bioactivity of BMP-2. Quite the opposite, as proven in Fig. 1I, the rise of native temperature could destroy hydrogen bond interactions and speed up hydrogel degradation to extend the discharge of BMP-2 and bone-inducing exercise [65].
The osteogenic impact in vitro. A: ALP staining. B: Alizarin purple staining. C: ALP exercise. D: Microscopic pictures of alizarin purple staining, purple: deposition of mineralized nodules, scale bar :100 μm
Toxicity and metabolism of Hydrogel + T8IC + Laser + BMP-2 + H2
O
2 in vivo
So as to monitor the metabolism of Hydrogel + T8IC + Laser + BMP-2 + H2O2 within the physique of BALB/C nude mice, an in vivo fluorescence detector with NIR-II fluorescence scanner (Determine S3) was designed and assembled. The emission spectrum of T8IC NPs belongs to NIR-II window, so the fluorescence scanner may detect the residual content material of T8IC NPs to observe the metabolism of Hydrogel + T8IC + Laser + BMP-2 + H2O2in vivo. The sol-gel transition of chitosan/β-glutamine/gelatin hydrogel occurred in 5 min when the temperature reached to 37 ℃ [15]. After the appliance of NIR-II phototherapy brokers (T8IC NPs and H2O2) and laser irradiation within the current research, the impact of PTT made the sol-gel transition time shorten to 2 min (45 ℃). Thus, when the hydrogel was utilized in vivo, it wouldn’t return to answer if temperature goes down from 45 ℃ to the physique temperature and it is ready to carry out managed launch of the medication. As proven in Fig. 6A, after 21 days of subcutaneous injection behind the mice and light-weight irradiation for as soon as, the hydrogel fashioned and saved steady. There was no apparent inflammatory response, implying no toxicity of hydrogels in vivo. The native temperature across the subcutaneous injection behind the mice raised with the time of laser irradiation (Fig. 6B). The focus of T8IC NPs in vivo decreased with time in each teams of Hydrogel + T8IC + Laser + BMP-2 + H2O2 and T8IC NPs (Fig. 6C). When mixed with the hydrogel, T8IC NPs in Hydrogel + T8IC + Laser + BMP-2 + H2O2 may very well be maintained with a better focus and longer time in comparison with T8IC NPs alone, which is helpful to the managed launch of medication. It was proved that the hydrogel offered with good biocompatibility and encapsulation skill.
The periodontitis mannequin was established on feminine C57BL/6 mice. Micro-CT was utilized to evaluate the institution of periodontitis across the maxillary second molar. Contemplating the attainable harm to the periodontal tissue generated from PDT and PTT, the remedy technique was as soon as every week by periodontal native hydrogel injection and laser irradiation. Determine 6D illustrated the institution of periodontitis and remedy progress. On one hand, remedy as soon as every week and 3 times in whole may depart time for the periodontal tissue to get well, scale back drug dosage and keep away from different attainable negative effects. Alternatively, it may get rid of periodontal pathogens and promote bone formation to the total extent. The physique weight of mice was repeatedly monitored through the 6-week periodontitis mannequin development and remedy. There have been no apparent modifications of the load after native injection of the hydrogel and laser irradiation (Fig. 6E, P > 0.05). The blood routing check and biochemical examination after remedy was performed to discover the impact on peripheral blood cells index. WBC, RBC, Hb, HCT and all of the indexes representing hepatorenal operate (together with TBIL, Alb, GLB, ALT, AST, ALP, GGT, BUN, CRE and UA) had no vital distinction between the experimental and management teams (Fig. 6F and G, P > 0.05), implying an excellent biosafety of Hydrogel + T8IC + Laser + BMP-2 + H2O2 in vivo.
Animal experiments. A: 0 d: the pre-gel was injected subcutaneously in nude mice, 21 d: domestically fashioned hydrogel and residual T8IC NPs had been marked by a inexperienced circle. B: The native physique temperature of mice after laser irradiation, min: minute. C: Focus modifications of T8IC NPs after subcutaneous injection, d: day. D: Schematic illustration of periodontitis mannequin development and remedy, blue: silk ligature with P. gingivalis, w: week. E: The modifications of physique weight in every group throughout remedy, w: week. F: The blood routine examination of mice after remedy. G: The hepatorenal operate of mice after remedy
Osteogenic and anti inflammatory effectsin vivo
The gap between cemento-enamel junction and the alveolar bone crest (CEJ-ABC) was measured to guage the periodontal regeneration [15]. The quantitative evaluation of CEJ-ABC confirmed that there was a major alveolar bone regeneration within the Hydrogel + T8IC + Laser, Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2.A greater bone restoration within the Hydrogel + T8IC + Laser + BMP-2 + H2O2 was noticed compared with Hydrogel + T8IC + Laser and Hydrogel + BMP-2 (Fig. 7A and B, P < 0.05). Lower within the bone mineral density (BMD, mg/cm3) and bone quantity fraction (bone quantity/tissue quantity, BV/TV, %) was detected after institution of the periodontitis mannequin within the experimental teams. Each BMD and BV/TV had been considerably highest within the Hydrogel + T8IC + Laser + BMP-2 + H2O2 among the many different 4 experimental teams (Fig. 7C and D, P < 0.05). All these outcomes indicated that hydrogel alone didn’t have the osteogenic impact and hydrogel mixed with T8IC + laser or BMP-2 may partially enhance the bone regeneration. Considerably, Hydrogel + T8IC + Laser + BMP-2 + H2O2 exhibited the perfect therapeutic impact.
Irritation is a type of necessary immune protection. When the tissue is threated by toxins or micro organism, the inflammatory response is initiated and it’s a regular state of self-protection and helpful to human well being. Nonetheless, irritation can be probably dangerous, if not handled in a well timed method, and can trigger cytokine storm and different bodily dysfunction [66]. The pathogenesis of periodontitis is expounded to the oral micro organism and the imbalance of immune and inflammatory responses. Below inflammatory situations, periodontal tissue and immune cells secrete quite a lot of cytokines (interleukin-1: IL-1, interleukin-6: IL-6, interleukin-10: IL-10, tumor necrosis issue α: TNF-α), which may promote the degradation of connective tissue and speed up bone destruction [67]. Anti-inflammation is equally necessary to the bacterial elimination within the remedy of infectious ailments. Delicate temperature PTT may inhibit the technology of proinflammatory cytokines (TNF-α, IL-6, IL-1β and IL-10) and the inflammatory stage was transformed into the regrowth of recent tissue [68, 69].
As proven in Fig. 7A and D, Hydrogel + T8IC + Laser + BMP-2 + H2O2 exhibited a greater osteogenic impact than Hydrogel + BMP-2 and Hydrogel + T8IC + Laser. Moreover, even with out the appliance of BMP-2, there was bone regeneration within the Hydrogel + T8IC + Laser, implying enhanced PDT and gentle PTT could induce bone regeneration by way of enhancing the anti-inflammation efficiency, as had been reported [58, 59]. To additional affirmation, immunohistochemical staining was carried out to analysize the inflammatory state of periodontal tissue. The expression of the proinflammatory cytokines (TNF-α, IL-6 and iNOS) was considerably greater within the group of Periodontitis and Hydrogel (Fig. 7E H). The cytokines within the Hydrogel + T8IC + Laser was decrease than that of Hydrogel + BMP-2 and there was a least inflammatory state within the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (P < 0.05). All these outcomes above implied that PTT and PDT generated by Hydrogel + T8IC + Laser and Hydrogel + T8IC + Laser + BMP-2 + H2O2 could promote bone regeneration by way of assuaging the irritation state.
Osteogenic and anti inflammatory results in vivo.A: Micro-CT photos of maxillary alveolar bone surrounding the maxillary second molars (M2) after the remedy. Inexperienced arrow reveals distance between cemento-enamel junction and the alveolar bone crest (CEJ-ABC). B: Quantitative evaluation of CEJ-ABC. C: Bone mineral density (BMD, mg/cm3). D: Bone quantity fraction (bone quantity/tissue quantity, BV/TV, %). E: Immunohistochemical staining, scale bar: 50 μm. F: IOD/space of IL-6. G: IOD/space of TNF-α. H: IOD/space of iNOS.
Biosafety monitoring
The hemolysis assay was carried out to detect the biosafety of T8IC NPs in vitro. No hemolysis of purple blood cells from mice incubated with totally different concentrations of T8IC NPs was noticed (Determine S4), which validated its biocompatibility. The physique weight of mice, blood routing check and biochemical examination had been additionally performed above to confirmed its biosafety. To additional confirm their potential toxicity, H&E staining of main organs (together with coronary heart, liver, spleen, lung and kidney) was additionally performed. The outcomes demonstrated that the cell morphology of main organs was intact and there was no apparent infiltration of inflammatory cells (Fig. 8). All outcomes above indicated the superb biosafety and histocompatibility of Hydrogel + T8IC + Laser + BMP-2 + H2O2.
Biocompatibility analysis after totally different remedies. H&E staining photos of main organs (coronary heart, liver, spleen, lung and kidney), scale bar: 100 μm
