Characterization of the HAABP injectable hydrogels

HA is a natural polymer in soft tissues with excellent biological activity, low immunogenicity, and tissue absorbability. It has been widely used in ophthalmology, intra-articular injection, and soft tissue healing.28,2928. M. F. Graça, S. P. Miguel, C. S. Cabral, and I. J. Correia, Carbohydr. Polym. 241, 11636 (2020). https://doi.org/10.1016/j.carbpol.2020.11636429. Z. Chen, F. Zhang, H. Zhang, L. Cheng, K. Chen, J. Shen, J. Qi, L. Deng, C. He, H. A. Santos, and W. Cui, Adv. Sci. 8, 2004793 (2021). https://doi.org/10.1002/advs.202004793 In this study, HA was chosen as the main polymer chain segment. To construct injectable antibacterial hydrogels, the HA–SH was synthesized in which the sulfhydryl groups could cross-link with Ag+. First, the characteristic peaks of the sulfhydryl group were detected on the 1H NMR (600M, Bruker AVANCE) spectra of HA–SH [Fig. 1(a)], indicating the successful modification of the sulfhydryl group on HA molecular chains, and the substitution rate was approximately 19%. The hyaluronic acid–Ag+ hydrogel (HAA) was formed by the coordination bonds between sulfhydryl and Ag+, while the coordination bonds constructed the HAABP hydrogel among sulfhydryl, Ag+, and BP, thus endowing HAA and HAABP hydrogels with injectable and self-healing features [Figs. 1(b)–1(d)]. The results showed that both hydrogels could be extruded from a needle by breaking the coordination bonds in response to an external force and maintained great extrusion homogeneity throughout, which was suited for irregular defects in clinical. When the external force was removed, the separated pieces of HAABP hydrogel would remix and regain excellent mechanical strength. The micromorphology of HAABP hydrogel after the cross-linking by the coordination bonds between Ag+ and SH was detected by (scanning electron microscope) SEM [Fig. 1(e)]. The results revealed that both HAA and HAABP hydrogels had the typical morphology of hydrogels with porous structures and smooth surfaces, which indicated that the addition of BP did not destroy the inherent structure of the HAABP hydrogels. As the previous study reported, the porous structure of hydrogel can promote the exchange of nutrients and the absorption of tissue exudates, thus facilitating wound healing.3030. B. Guo, R. Dong, Y. Liang, and M. Li, Nat. Rev. Chem. 5, 773 (2021). https://doi.org/10.1038/s41570-021-00323-z Moreover, at high magnification of SEM images, sheet structures could be observed on the surface of HAABP hydrogel, while no sheet structures could be observed on the surface of HAA hydrogel, which further suggested the successful introduction of BP.To further verify the interaction between BP and Ag+, the 2D morphology of BP adsorbing Ag+ was detected by TEM. As shown in Fig. 1(f), the bare BP nanosheets possessed smooth surface structure and sharp edges with an average diameter of 174.5 ± 23.8 nm, similar to the range reported.3131. Z. Wang, J. Zhao, W. Tang, L. Hu, X. Chen, Y. Su, C. Zou, J. Wang, W. Lu, W. Zhen, R. Zhang, D. Yang, and S. Peng, Small 15, 1901560 (2019). https://doi.org/10.1002/smll.201901560 After the electrostatic absorption of Ag+, the morphology of BP became blunt and thickened, which was also verified by the element mapping [Fig. 1(f)]. In addition, the average diameter of [email protected] showed little change compared with BP nanosheets (P −1, respectively (Fig. S1), which were following the previous studies.1515. L. Cheng, Z. Chen, Z. Cai, J. Zhao, M. Lu, J. Liang, F. Wang, J. Qi, W. Cui, and L. Deng, Small 16, 2005433 (2020). https://doi.org/10.1002/smll.202005433 After the absorption of Ag+, [email protected] nanosheets exhibited a remarkable decrease in all characterized peaks, which confirmed the successful introduction of Ag+.The mechanical properties of HAABP were examined by rheometer [Figs. 2(a)–2(d)]. Four experimental groups were set according to different concentrations of BP as HAABP-1 (10 μg/ml BP), HAABP-2 (50 μg/ml BP), HAABP-3 (100 μg/ml BP), and HAABP-4 (200 μg/ml BP), and HAA was considered as the control group. All hydrogels formed a solid with a higher elastic modulus value (G′) in the initial stage. As the strain increased, the value of loss modulus (G′′) increased step by step and became higher than G′, illustrating the hydrogels in the formation of liquid. With the increase in BP concentration, the critical G′ value increased, and the G′ values of different groups were 785.66 (control), 850.16 (HAABP-1), 908.34 (HAABP-2), 1115.87 (HAABP-3), and 1387.60 Pa (HAABP-4). For HAABP-4, the addition of black phosphorus nanosheets increased the shear modulus of HAA hydrogel by 1.76-fold, indicating that BP nanosheets formed co-coordination with HA–SH–Ag and that the dynamic coordination of silver ions with sulfhydryl groups on HA and phosphate groups on the surface of BP. In addition, compared with the conventional single coordination of Ag+, Cu2+, and Sr2+ and BP nanosheets,32–3432. F. Deng, P. Wu, G. Qian, Y. Shuai, L. Zhang, S. Peng, C. Shuai, and G. Wang, Nanotechnology 33, 245708 (2022). https://doi.org/10.1088/1361-6528/ac5aee33. J. Zeng, X. Geng, Y. Tang, Z.-C. Xiong, Y.-J. Zhu, and X. Chen, Chem. Eng. J. 437, 135347 (2022). https://doi.org/10.1016/j.cej.2022.13534734. X. Wang, J. Shao, M. Abd El Raouf, H. Xie, H. Huang, H. Wang, P. K. Chu, X. F. Yu, Y. Yang, A. M. AbdEl-Aal, N. H. M. Mekkawy, R. J. Miron, and Y. Zhang, Biomaterials 179, 164 (2018). https://doi.org/10.1016/j.biomaterials.2018.06.039 the HAABP hydrogel system provides a new method for the subsequent construction of BP-based biomaterials and the enhancement of mechanical properties. Then, the HAABP-3 was selected for the next test because of its appropriate intensity and low toxic sides [Fig. 2(b)].3535. K. Huang, J. Wu, and Z. Gu, ACS. Appl. Mater. Interfaces 11, 2908 (2019). https://doi.org/10.1021/acsami.8b21179 There was no difference in viscosity between HAA and HAABP-3, and with the increase in the shear rate, the viscosity decreased almost linearly [Fig. 2(c)], which further demonstrated the injectability of the HAA and HAABP-3 hydrogels. In addition, cyclic step-strain measurements were employed to detect the recoverability and re-healing of HAA and HAABP hydrogels under the high strains [Fig. 2(d)]. Both hydrogels maintained colloidal shapes before suffering a high shear rate, holding G′ values at 767.79 Pa (HAA) and 1128.97 Pa (HAABP-3). The G′ values fell to ≈18.55 (HAA) and ≈22.43 Pa (HAABP-3) when the strain was performed due to the destroyed internal networks. However, when the strain disappeared, the G′ values fully recovered within seconds. All these results suggested that HAABP hydrogel had excellent mechanical properties and injectable ability. Moreover, both hydrogels were immersed in phosphate buffer saline (PBS) to investigate their degradation behaviors and Ag+ releasing. As presented in Fig. 2(e), the hydrogels lost about 10% in quality on the first day. Then, significant degradation occurred in the following days, with HAA achieving complete degradation on day 8 and HAABP on day 10. The degradation rate of the ideal biomaterial must match the regeneration process of the tissue; if the degradation is too fast, it is not enough to support the inward growth of the tissue, and it is too slow, degradation will hinder the normal regeneration process of the tissue and lead to local fibrosis.3636. D. R. Griffin, W. M. Weaver, P. O. Scumpia, D. Di Carlo, and T. Segura, Nat. Mater. 14, 737 (2015). https://doi.org/10.1038/nmat4294 Compared with traditional hydrogel dressings or bio-papers, the degradation rates of HAA and HAABP hydrogels match the regeneration process of soft tissue wounds (1–2 weeks).33,3733. J. Zeng, X. Geng, Y. Tang, Z.-C. Xiong, Y.-J. Zhu, and X. Chen, Chem. Eng. J. 437, 135347 (2022). https://doi.org/10.1016/j.cej.2022.13534737. Y. Liang, J. He, and B. Guo, ACS Nano 15, 12687 (2021). https://doi.org/10.1021/acsnano.1c04206 The cumulative releasing of Ag+ in HAA arrived at 57% on day 1 and continued to increase to 80% on day 7, which was higher than that in HAABP [Fig. 2(f)]. Based on these results, the introduction of BP could prolong the degradation of HA hydrogels and delay the release of Ag+, which matches the early needs of infected wound healing.

In addition, the injectable force of the hydrogel was further evaluated by using an Instron tester at a speed of 5 mm/min to determine the force required to inject the hydrogel with different diameter needles (20, 22, 25, and 27G). In Fig. S2, the injection force of HAA hydrogel to 20, 22, 25, and 27 G needles was 2.98 ± 0.10, 10.27 ± 0.34, 12.62 ± 0.21, and 25.93 ± 0.43 N, respectively. However, the injection force of HAABP hydrogel with black phosphorus was significantly increased to 3.83 ± 0.09, 11.21 ± 0.18, 14.15 ± 0.79, and 27.30 ± 0.55 N, respectively. This result shows that both HAA and HAABP hydrogel have excellent injectability and further prove that black phosphorus enhances the interaction of thiol groups with silver ions. Moreover, when the 20 G injection needle was used, the injection forces of HAA and HAABP injectable hydrogels were 2.98 ± 0.10 and 3.83 ± 0.09 N, respectively, which were in accordance with ISO 7886-1:2017. However, when the diameter of the injection needle is smaller, it needs to exceed the ISO standard injection force, which requires further improvement in the later stage to meet wider and more complex clinical applications.

The photothermal effect and antibacterial properties of the HAABP hydrogel

Because of the photothermal-mediated antibacterial ability of BP, we assessed the photothermal effect of HAABP hydrogel irradiated under NIR (808 nm) before evaluating the antibacterial activity. As presented in Figs. 3(a) and 3(d), with the increasing exposure of NIR, HAABP hydrogel showed a gradually increasing photothermal effect with a plateau of temperature (45 °C) at about 120 s, reaching the bacteria-killing conditions. No significant temperature change was observed in HAA. The results indicated the potential photothermal applications of HAABP hydrogel. Subsequently, the antibacterial properties of HAABP hydrogel under NIR against the gram-positive S. aureus were assessed through an inhibition ring test and live/dead staining of bacteria. It was well known that Ag+ had a broad-spectrum inhibitory effect on the proliferation of both gram-negative and gram-positive bacteria by interfering with protein biosynthesis in bacteria.2323. L. X. Yin, J. Zhang, I. S. Zhao, M. L. Mei, Q. Li, and C. Chu, Int. J. Nanomed. 15, 2555 (2020). https://doi.org/10.2147/IJN.S246764 In this study, HA hydrogels also showed the inhibitory function of bacterial proliferation due to the presence of Ag+. After 24 h of co-culture, the inhibition area of HAA hydrogel reached 2.223 ± 0.033 cm2 [Fig. 3(b)]. Compared to the control group, HAA hydrogel showed better antibacterial ability in fluorescent staining. The two-dimensional structure of BP had a direct bactericidal ability due to the sharp edge.3838. T. Guo, S. Zhuang, H. Qiu, Y. Guo, L. Wang, G. Jin, W. Lin, G. Huang, and H. Yang, Part. Part. Syst. Charact. 37, 2000169 (2020). https://doi.org/10.1002/ppsc.202000169 In this study, it was found that the inhibition area of HAABP hydrogel reached 2.276 ± 0.024 cm2 (P > 0.05 vs HAA hydrogel). The main reason might be that BP and Ag+ were closely connected in HA, and the degradation product of BP was the released phosphate instead of directly BP nanosheets. As expected, after NIR irradiation, HAABP hydrogel (HAABP–NIR) exhibited the best antimicrobial activity (2.639 ± 0.069 cm2) (P Fig. 3(e)].In short, all these results confirmed the antibacterial property in vitro of HAABP hydrogel. Furthermore, the antibacterial effect was explored with live/dead staining, which labeled living bacteria with green fluorescence by SYTO9 and dead bacteria with red fluorescence by propidium iodide (PI). It was found that S. aureus in the control group showed remarkable survival, while in the HAA group, only faint green fluorescence [(4.180 ± 0.316) × 103/cm2] was detected [Figs. 3(c) and 3(f)]. The green fluorescence became less in the HAABP hydrogel [(3.165 ± 0.158) × 103/cm2], and the HAABP–NIR hydrogel presented the weakest green fluorescence [(1.242 ± 0.1722) × 103/cm2]. The results suggested the HAABP hydrogel synergistic antibacterial effect on S. aureus. Therefore, all results suggested that the HAABP hydrogel possessed an excellent photothermal effect. The converted temperature reached about 45 °C, and the bacteria's enzymes would be denatured and inactivated, thus leading to the death of bacteria. Moreover, the controlled photothermal effect and physiologic degradation made BP a better photothermal agent than Au or other nanomaterials.

HAABP hydrogel accelerated the regeneration of infected wound defects

The theoretical healing process in vivo and the mechanism of HAABP hydrogel accelerating the regeneration of infected wound defects were shown in Fig. 4(a). The anti-bacteria and regeneration-promoting abilities of HAABP–NIR hydrogel in vivo were evaluated by treating rats with a diameter of 1 cm full-thickness infected wound defect. At each time point (postoperative days 0, 3, 7, 10, and 14), we observed and recorded the morphology of wounds by a digital camera [Figs. 4(b) and 4(c)] to dynamically measure the wound healing rate. On day 3, both HAA and HAABP hydrogels exhibited similar wound repair rates (47.87 ± 1.60% and 47.44 ± 0.89%), which were higher than the control group (39.38% ± 0.69%) [Fig. 4(d)]. In the control group, the yellow pus dispersed around the wound sites, and the wounds were still wet without obvious crust-like tissue formed and signs of recovery until day 10. However, less yellow pus and degraded hydrogels were observed in groups of hydrogels on day 3. With the help of NIR, the wound appeared dryer and smaller in the HAABP–NIR hydrogel group (69.16% ± 1.44%), which suggested infection had been blocked effectively and repair proceeded in an orderly manner. With the extension of time, all groups tended to heal, but the healing rate in the control group seemed to increase slowly, especially in the first 10 days. Conversely, due to the released Ag+ and NIR-assisted thermal effect in the HAABP–NIR group, the defect was almost recovered on day 10 (86.44% ± 0.75%) and a linear scar appeared on day 14 (94.17% ± 0.89%). These exciting results will push the promising application of HAABP–NIR hydrogel for critical infected wound defect repair. The injectable property gave the hydrogel better applicability, especially in periprosthetic soft tissue defects.Wound healing was a highly spatiotemporally regulated process.4141. Z. Cai, Q. Saiding, L. Cheng, L. Zhang, Z. Wang, F. Wang, X. Chen, G. Chen, L. Deng, and W. Cui, Bioact. Mater. 6, 4506 (2021). https://doi.org/10.1016/j.bioactmat.2021.04.039 We also performed hematoxylin-eosin (HE), Masson, and immunofluorescent staining to investigate the healing process further. As shown in Fig. 5, the epidermis structures and regenerated hair follicles were observed in all groups after 7 days. Compared to the control group, the groups of different hydrogels, especially the HAABP–NIR group, recruited more fibroblasts and regenerated more newly born capillaries, which were vital for granulation maturity and wound contraction. On day 14, the unmatured tissues presented in the HAABP–NIR group were almost replaced with intact re-epithelialization, whereas the resultant epidermis in control and other hydrogel groups was thinner and immature. In the HAABP–NIR hydrogel group, the best anti-bacteria and re-regeneration abilities were observed due to the NIR-assisted photothermal effect of BP. The NIR was biocompatible to normal tissues and could penetrate deeper tissues for remote sterilization. Moreover, it had been reported that mild heat could promote the regeneration of blood vessels, which was beneficial for nutrition-oxygen supply and waste diffusion, and further facilitated the healing process by inhibiting the HSP70/NLRP3 pyroptosis signaling pathway in cells.42,4342. E. Chen, D. Xue, W. Zhang, F. Lin, and Z. Pan, FEBS Lett. 589, 4088 (2015). https://doi.org/10.1016/j.febslet.2015.11.02143. M. Li, S. Fuchs, T. Böse, H. Schmidt, A. Hofmann, M. Tonak, R. Unger, and C. J. Kirkpatrick, Tissue Eng., Part C 20, 328 (2014). https://doi.org/10.1089/ten.tec.2013.0087 However, the exact mechanisms remained to be discovered by further studies.Additionally, the immunofluorescent staining for assessing collagen deposition (Col I), angiogenesis (CD31), and inflammation expression (TNF-α) was also accomplished. Col I played a key role in the remodeling phase of wound healing. As shown in Figs. 6(a) and 6(b), the Col I expression increased in all groups from day 7 to day 14. All groups of hydrogels exhibited more deposition of Col I than the control group because the released Ag+ inhibited the negative effects caused by bacteria, and the process of self-repair was accelerated in all groups of hydrogels. However, the difference between HAA and HAABP hydrogel group was not statistically significant, and the main reason could be that the BP in the HAABP hydrogel could not promote healing. After the irradiation of NIR, the highest expression of Col I was detected in the HAABP–NIR group on day 14. The results verified that the nanomaterial BP with the photothermal effect could accelerate the regeneration process through the excellent PTT-assisted antibacterial ability under the irradiation of NIR. In addition, the photothermal effect could also promote the invasion of blood vessels to the wound sites and improve the supply of nutrition.4343. M. Li, S. Fuchs, T. Böse, H. Schmidt, A. Hofmann, M. Tonak, R. Unger, and C. J. Kirkpatrick, Tissue Eng., Part C 20, 328 (2014). https://doi.org/10.1089/ten.tec.2013.0087 As expected, the expression of the important indicator of vascularization CD31 in the HAABP–NIR group was highest on day 14. The fluorescent intensity of CD31 in HAA and HAABP was similar, and the control group showed the lowest expression of CD31 [Figs. 6(a) and 6(c)]. Moreover, TNF-α was used to assess the efficacy of the HAABP–NIR hydrogel in preventing infection. There was a high expression of TNF-α in the control group and a lower TNF-α expression in the HAA and HAABP hydrogel groups, while the lowest in the HAABP–NIR group owing to the synergetic bacteriostatic ability of Ag+ and PTT (Fig. S5). Hence, by simultaneously upregulating the expression of CD31 to accelerate angiogenesis, improving the deposition of Col I, and decreasing the production of TNF-α to reduce the inflammatory response, HAABP–NIR hydrogel effectively promoted the wound healing process.