Synthesis and characterizations of Hydrogel + T8IC + Laser + BMP-2
T8IC is a novel organic semiconducting material, mainly used in high performance organic photovoltaic device. T8IC nano-particles (T8IC NPs) were synthesized through reprecipitation. The Zeta potential of T8IC NPs was 21.9 ± 1.12 mV. It was homogeneously distributed and kept stable in PBS and DMEM for 14 days (Figure S1A). The absorption spectrum of T8IC NPs was 600 ~ 900 nm (Figure S1B). It belonged to the second near-infrared region spectrum (NIR-II, 900 ~ 1700 nm), of which the emission fluorescence spectrum was 900 ~ 1100 nm (Figure S1C). The TEM image exhibited spherical morphology of T8IC NPs (Figure S1D). The average diameter of T8IC NPs was about 160 nm (Figure S1E-S1F). Besides, T8IC NPs could produce ROS after 808 nm laser irradiation, suggesting a good potential for PDT(40). Both PTT and PDT of T8IC NPs exhibited good tumor elimination when combined with sorafenib in our previous study(40).
The translucent pre-gel was injectable and flowed like a viscous liquid. Interestingly, the sol-gel transition occurred when the temperature was around 45 ℃ within 4 minutes (Fig. 1A-1B). Scanning electron microscope (SEM) images revealed porous structure within the freeze-dried hydrogel (Fig. 1C), which facilitated the storage and release of drugs and cell migration(39). The rheological properties of Hydrogel + T8IC + Laser + BMP-2 was investigated through rheological tests. The strain dependent oscillatory rheology experiments (Fig. 1D) revealed that the storage modulus (G') and the loss modulus (G'') remained constant when strain was below 100% while the frequency was 1 Hz. Furthermore, the G' was over 100 Pa and much higher than the G'', indicating stable hydrogel networks formed. In addition, the viscosity of hydrogel decreased sharply with increasing shear rate (Fig. 1E), implying a shear thinning property, which is beneficial for further injection(42). The temperature dependent oscillatory rheology experiment (Fig. 1F) was also conducted and when the temperature was below 44.69 ℃, the value of G' and G'' was stable and G'' was higher than that of G'. While modulus of G' and G'' increased with the temperature sharply and the G' and G'' crossed at a temperature of 44.69 ℃. Then, the value of G' was higher than that of G'', implying the sol-gel transition. The weight loss (%) of Hydrogel + T8IC + Laser + BMP-2 increased with time at room temperature (24 ℃) and 37 ℃ (Fig. 1G). 82% weight loss at room temperature (24 ℃) after 10 days and 93% weight loss at 37 ℃ after 14 days. In order to evaluate the effective laser (808 nm) depth penetration of the hydrogel, the residual laser intensity was detected along with the hydrogel depth (Fig. 1H). The laser intensity decreased with the hydrogel depth. 62% residual laser intensity was detected when the hydrogel depth was 20 mm, indicating that hydrogel combined with T8IC NPs was capable for the treatment of periodontitis with deep periodontal pocket. The BMP-2 release from the hydrogel (Fig. 1I) had been monitored. Cumulative BMP-2 release of Hydrogel + T8IC + Laser + BMP-2 was higher than that of Hydrogel + T8IC + BMP-2.
A good photothermal conversion ability of Hydrogel + T8IC + Laser + BMP-2 was detected under different concentrations of T8IC NPs and different laser power (808 nm) (Fig. 1J-1K). The photothermal effect was concentration and power dependent. It was also presented with a good photothermal stability (Fig. 1L).
Photothermal effects on the cell proliferation in vitro
In this study, the cytotoxicity of T8IC NPs was assessed through CCK-8 assay. The cell viability of MC3T3-E1 cells wasn’t influenced by T8IC NPs when the concentration was under 50 µg/ml without irradiation (Fig. 2A), implying a good biosafety. As for the photothermal effects, Hydrogel + T8IC + Laser + BMP-2 exhibited remarkable 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 effect of Hydrogel + T8IC + Laser + BMP-2 was also monitored in 96-well plates (Fig. 2C). The temperature of Hydrogel + T8IC + Laser + BMP-2 raised to 45 ℃ within 4 minutes, while it was only up to 28.6 ℃ in the Hydrogel + Laser group. The live/dead cell staining also proved that the higher temperature was and more red-fluorescence detected, implying less cells alive (Fig. 2D). Thus, a relative mild temperature should be chosen to protect cells from the hyperthermia and also meet the need of bactericidal effect in the subsequent study.
Antibacterial effect in vitro
With high prevalence of antibiotic-resistant bacteria, many novel nano-material based antibacterial strategies has been created at the right time(43). However, the side effect of high temperature created by PTT on the surrounding tissue may limit its clinical application. Thus, it is meaningful to seek a mild temperature which can kill the bacterial and also reduce the damage to the surrounding tissue.
Firstly, we assessed the temperature on the bacterial inhibition. The hydrogel and Hydrogel + BMP-2 had no effect on the P. gingivalis proliferation (Fig. 3A). The bacterial number decreased at a controlled temperature of 37℃ after laser irradiation, indicating that PDT plays an important role in the bacterial ablation. Besides, the eradication rates of pathogens increased with the temperature. The antibacterial activity of Hydrogel + T8IC + Laser + BMP-2 (45℃) was enhanced compared with Hydrogel + T8IC + Laser + BMP-2 (37℃), but still not enough to completely eliminate biofilms. Thus, we planned to promote the PDT to facilitate the antibacterial effect instead of increasing the temperature, which was harmful to the cell proliferation. Occasionally, H2O2 was found to be a good photosensitizer, which was commonly used as periodontal pocket rinse solution after periodontal initial therapy. Although H2O2 can effectively kill bacteria by attacking and destroying DNAs and proteins, the high concentration of H2O2 exhibited toxic effect to normal tissue. After catalyzed into reactive oxygen species (ROS), H2O2 manifested better antibacterial efficiency(19, 44). Therefore, a lower concentration of H2O2 and enhanced antibacterial efficiency was designed. DBPF probe was used to assess changes of ROS content in Hydrogel + T8IC + Laser + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 under 808 nm laser irradiation. As shown in Fig. 3B, the ROS yield was calculated by monitoring the oxidation of DPBF at 418 nm in dichloromethane(45). The more DBPF loss after adding H2O2, implying more ROS generated and a better PDT effect. Other studies had shown that the combination of PDT and PTT could destroy the bacterial membranes and enhance bacterial eradication(36, 46, 47). As a result, the antibacterial effect had been promoted in the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45℃) group compared to the Hydrogel + T8IC + Laser (45 ℃) group (Fig. 3C), suggesting that synergistic effect of an enhanced PDT and a mild temperature PTT (45 ℃) could acquire a better bacterial eradication. There was no significant difference among control, Hydrogel, Hydrogel + H2O2 and Hydrogel + BMP-2 after 10 days culture by the colony forming unit assay (Fig. 3D). Both Hydrogel + T8IC + Laser (45℃) and Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45℃) had significantly less P. gingivalis colonies, while the pathogens in the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45℃) were nearly entirely eliminated. In addition, P. gingivalis suspended within the BHI medium, so 3D images of the live/dead (green/red) bacterial fluorescence staining were built up to detect the biofilm viability of P. gingivalis (Fig. 3E). Absolutely only green fluorescence was observed in the group of control, Hydrogel, Hydrogel + H2O2 and Hydrogel + BMP-2, indicating biofilms were intact. A small amount of red fluorescence was detected in Hydrogel + T8IC + Laser (45℃) and the biofilms were almost stained red in Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45℃), consistent with the colony forming unit assay. In general, the phototherapy generated from Hydrogel + T8IC + Laser + BMP-2 + H2O2 including enhanced PDT and mild PTT, exhibited a superior bactericidal effect. Therefore, we finally selected 45 ℃ as a mild temperature in the following study.
Osteogenic effect in vitro
In order to detect the effect of enhanced PDT and mild PTT to the cell proliferation, the cell spreading of MC3T3-E1 cells after different treatments was monitored. As shown in Fig. 4A, Hydrogel, Hydrogel + H2O2 and Hydrogel + BMP-2 had less effect on the actin filaments labelled by phalloidine (red), while cell shrinkage and detachment in the Hydrogel + T8IC + Laser (45 ℃) and Hydrogel + T8IC + Laser + BMP-2 + H2O2 (45℃) were observed due to the combined effects of PTT and PDT. Delightfully, the cell regained the bioactivity, cell spreading and attachment after another 24-hour culture. Besides, although the proliferation of MC3T3-E1 cells was inhibited in Hydrogel + T8IC + Laser and Hydrogel + T8IC + Laser + BMP-2 + H2O2 1 day after treatment, the cell viability had resuscitated 3 days after treatment (Fig. 4B).
Although BMP-2 enhances the recruitment and angiogenesis of osteoblast precursor cells and has attracted much attention for its excellent bone-inducing ability(27, 28), its commercial use is limited by two major aspects: firstly, BMP-2 has a short half-life (only 7 minutes) in the physiological environment and a high dose of BMP-2 is required to be loaded into those scaffolds to solve its instability and rapid inactivation, which may result in significant costs and an increased risk of side effects, including inflammation, nerve damage, ectopic ossification and tumorigenesis(48–50). Secondly, absorbable collagen sponge and calcium phosphate(51, 52) are currently the only two BMP-2 carriers approved for clinical use. However, because of the low affinity of these vectors for BMP-2 and their low retention rate, the use of high doses can also exacerbate local and systemic adverse effects. To overcome these problems and optimize the bone healing process, various approaches have focused on improving the loading rate of BMP-2 while maintaining its biological activity. Among them, injectable hydrogels with 3D networks have become the primary choice for BMP-2 carriers in tissue engineering due to their high water-absorption, injectable ability, encapsulation ability, biocompatibility and biodegradability(29–31).
Osteoblast differentiation was assessed by mRNA expression (Figure S2), quantitative analysis of ALP activity, ALP staining and alizarin red staining. After 4 days culture, gene expression of osteogenic mRNAs (early osteogenic marker: ALP, specific osteogenic differentiation markers in the earlier stage: Runx2, late osteogenic marker: OCN, non-collagenous proteins: OPN, extracellular matrix protein: Col-I) was much higher in the Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 (Fig. 4C-G, P < 0.05). While the mRNA expression (ALP, OCN and OPN) of Hydrogel + T8IC + Laser + BMP-2 + H2O2 was significantly higher than that of Hydrogel + BMP-2 (P < 0.05), indicating that PTT could induce the release of BMP-2 and promote osteogenic gene expression, as shown in Fig. 1H.
The ALP staining (Fig. 5A) and alizarin red staining (Fig. 5B) was consistent with the results of PCR. Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 exhibited intensified staining of ALP and mineralized nodules. In addition, the ALP activity of Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 was much higher than other groups after both 4 days and 7 days culture (Fig. 5C, P < 0.05). The ALP activity of Hydrogel + T8IC + Laser + BMP-2 + H2O2 after 7 days culture was higher than that of Hydrogel + BMP-2. Furthermore, the microscopic photographs of alizarin red staining showed there was highest density of mineralized nodules deposition in the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (Fig. 5D). All those results proved that the mild PTT and enhanced PDT generated by Hydrogel + T8IC + Laser + BMP-2 + H2O2 may not affect the release and bioactivity of BMP-2. On the contrary, as shown in Fig. 1H, the increase of local temperature may destroy hydrogen bond interactions and accelerate hydrogel degradation to increase the release of BMP-2 and bone-inducing activity (53).
Toxicity and metabolism of Hydrogel + T8IC + Laser + BMP-2 + H 2 O 2 in vivo
In order to monitor the metabolism of Hydrogel + T8IC + Laser + BMP-2 + H2O2 in the body of BALB/C nude mice, an in vivo fluorescence detector with NIR-II fluorescence scanner (Figure S3) was designed and assembled. The emission spectrum of T8IC NPs belongs to NIR-II window, so the fluorescence scanner could detect the residual content of T8IC NPs to monitor the metabolism of Hydrogel + T8IC + Laser + BMP-2 + H2O2in vivo. After 21 days of subcutaneous injection in the back of the mice, the hydrogel formed and there was no obvious inflammatory response (Fig. 6A), implying no toxicity of hydrogels in vivo. The local temperature around the subcutaneous injection in the back of the mice raised with the time of laser irradiation (Fig. 6B). The concentration of T8IC NPs in vivo decreased with time in both groups of Hydrogel + T8IC + Laser + BMP-2 + H2O2 and T8IC (Fig. 6C). When combined with the hydrogel, T8IC NPs in Hydrogel + T8IC + Laser + BMP-2 + H2O2 could be maintained with a higher concentration and longer time compared to T8IC NPs alone, which is beneficial to the controlled release of drugs. It was proved that the hydrogel presented with good biocompatibility and encapsulation ability.
The periodontitis model was established on female C57BL/6 mice. Micro-CT was applied to assess the establishment of periodontitis around the maxillary second molar. Considering the possible damage to the periodontal tissue generated from PDT and PTT of T8IC + Laser, the treatment strategy was once a week by local hydrogel injection and laser irradiation. Figure 6D illustrated the establishment of periodontitis and treatment progress. On one hand, treatment once a week and three times in total could leave time for the periodontal tissue to recover, reduce drug dosage and avoid other possible side effects. On the other hand, it could eliminate periodontal pathogens and promote bone formation to the full extent. The body weight of mice was continuously monitored during the 6-week periodontitis model construction and treatment. There were no obvious changes of the weight after local injection of the hydrogel and laser irradiation (Fig. 6E, P > 0.05). The blood routing test and biochemical examination after treatment was conducted to explore the effect on peripheral blood cells index. WBC, RBC, Hb, HCT and all the indexes representing hepatorenal function (including TBIL, Alb, GLB, ALT, AST, ALP, GGT, BUN, CRE and UA) had no significant difference between the experimental and control groups (Fig. 6F-6G, P > 0.05), implying a good biosafety of Hydrogel + T8IC + Laser + BMP-2 + H2O2in vivo.
Osteogenic and anti-inflammatory effects in vivo
The distance between cemento-enamel junction and the alveolar bone crest (CEJ-ABC) was measured to evaluate the periodontal regeneration(39). The quantitative analysis of CEJ-ABC showed that there was a significant alveolar bone regeneration in the Hydrogel + T8IC + Laser, Hydrogel + BMP-2 and Hydrogel + T8IC + Laser + BMP-2 + H2O2 and a better bone recovery in the Hydrogel + T8IC + Laser + BMP-2 + H2O2 was observed when compared with Hydrogel + T8IC + Laser and Hydrogel + BMP-2 (Fig. 7A-7B, P < 0.05). Decrease in the bone mineral density (BMD, mg/cm3) and bone volume fraction (bone volume/tissue volume, BV/TV, %) was detected after establishment of the periodontitis model in the experimental groups. Both BMD and BV/TV were significantly highest in the Hydrogel + T8IC + Laser + BMP-2 + H2O2 among the other four experimental groups (Fig. 7C-7D, P < 0.05). All those results indicated that hydrogel alone didn’t have the osteogenic effect and hydrogel combined with T8IC + laser or BMP-2 could partially improve the bone regeneration. Significantly, Hydrogel + T8IC + Laser + BMP-2 + H2O2 exhibited the best therapeutic effect.
Inflammation is a kind of important immune defense. When the tissue is threated by toxins or bacteria, the inflammatory response is initiated and it is a normal state of self-protection and beneficial to human health. However, inflammation is also potentially harmful, if not dealt with in a timely manner, and will cause cytokine storm and other physical dysfunction(54). The pathogenesis of periodontitis is related to the oral bacteria and the imbalance of immune and inflammatory responses. Under inflammatory conditions, periodontal tissue and immune cells secrete a variety of cytokines (interleukin-1: IL-1, interleukin-6: IL-6, interleukin-10: IL-10, tumor necrosis factor α: TNF-α), which could promote the degradation of connective tissue and accelerate bone destruction(55). Anti-inflammation is equally important to the bacterial elimination in the treatment of infectious diseases. Mild temperature PTT could inhibit the generation of proinflammatory cytokines (TNF-α, IL-6, IL-1β and IL-10) and the inflammatory stage was converted into the regrowth of new tissue(56, 57).
As shown in Fig. 7A-7B, even without the application of BMP-2, there was bone regeneration in the Hydrogel + T8IC + Laser, implying enhanced PDT and mild PTT may induce bone regeneration through enhancing the anti-inflammation performance, as had been reported46,47. To further confirmation, immunohistochemical staining was performed to analysis the inflammatory state of periodontal tissue. The expression of the proinflammatory cytokines (TNF-α, IL-6 and iNOS) was significantly higher in the group of Periodontitis and Hydrogel (Fig. 7E-7H). The cytokines in the Hydrogel + T8IC + Laser was lower than that of Hydrogel + BMP-2 and there was a least inflammatory state in the Hydrogel + T8IC + Laser + BMP-2 + H2O2 (P < 0.05). Furthermore, Hydrogel + T8IC + Laser + BMP-2 + H2O2 exhibited a better osteogenic effect than Hydrogel + BMP-2 and Hydrogel + T8IC + Laser. All those results above confirmed that PTT and PDT generated by Hydrogel + T8IC + Laser and Hydrogel + T8IC + Laser + BMP-2 + H2O2 may promote bone regeneration through alleviating inflammation state.