3.1 Physical and Chemical Characterization of Hydrogel
It was found by accident that a layer of PVA solution covered on BC could keep the shape and structure of BC under low temperature. After freeze-melt cycle, no PVA covered BC exhibited collapse form and internal water flowed out, while PVA covered BC formed a two-layer hydrogel by physical cross-linking (Fig. S1). Based on this discovery and inspired by the philosophy of “Tai Chi”, we performed functional modifications around BC while maintaining the structure and property of native BC.
The synthesis procedure of Yang and Tai-Chi hydrogel was shown in Fig. S2. Dopamine hydrochloride (DA) was oxidized and polymerized into polydopamine in alkaline solution and uniformly dispersed in polyvinyl alcohol (PVA) solution. DA oxidation made the PVA solution darker (Fig. S3). The UV-vis spectroscopy analysis demonstrated that Yang solution with PDA have an absorption peak at 320 nm, and the peak value was positively correlated with DA concentration, which is the π-π* transition of aromatic hydrocarbons containing benzene ring structure (Fig. S4). Elemental analysis results showed that with the addition of DA, N element could be detected in the dried Yang hydrogel (Table S3). The above results indicated that DA was successfully oxidized and polymerized into PVA solution to form Yang solution and hydrogel containing PVA and PDA. The transformation process of sol-to-gel of Yang was visualized in Fig. S5. Cross-sectional FSEM images Tai-Chi hydrogel exhibited that Yin and Yang were crosslinked at the boundary between Yin and Yang hydrogel, and the rest of Yin/Yang hydrogel still maintained their natural network structure (Fig. 2a). This indicated that peripheral Yang hydrogel maintained the fiber morphology and structure of native BC under low temperature condition.
We observed that Yang hydrogel could withstand various deformation, including compression, stretching and knotted stretching (Fig. 2b). Similarly, Tai-Chi hydrogel exhibited good stretchability, including horizontal stretch and four-side stretch (Fig. 2c), and Yang hydrogel was seen to stretch more than Yin hydrogel during stretching process (Fig. 2d). Tensile test results demonstrated that DA addition had no significant effect on mechanical property of Yang hydrogel. The maximum elongation of Yang hydrogel with various concentrations of DA was from 214.39 ± 19.42% to 257.06 ± 10.11%, the tensile strength was from 201.35 ± 26.26 to 227.51 ± 15.15 KPa, the Young's modulus was from 23.88 ± 2.20 KPa to 29.00 ± 3.68 KPa. However, Yin hydrogel (BC) displayed significantly lower maximum elongation (27.93 ± 5.27%), higher tensile strength (1.71 ± 0.16 MPa) and Young’s modulus (5.54 ± 1.68 MPa) as reported before (Fig. 2e-h) [13], which indicated that Yang hydrogel was softer and easier to stretch to fit skin movement. We observed that Tai-Chi hydrogel could be well attached on the skin and could move with finger joint movement (movie 1). In addition, we performed tensile test on the Yin-Yang boundary of the Tai-Chi hydrogel, which presented that the Yin-Yang boundary bonded firmly, and the tensile fracture was in the Yang hydrogel part (movie 2), that may be related to the infiltration of the Yang solution into Yin hydrogel network to form a double-network hydrogel (Fig. 2a (iii)) to improve the tensile strength than Yang hydrogel.
3.2 Photothermal Characterization of Hydrogel
As shown in Fig. S6, the addition of dopamine could rapidly warm the Yang hydrogel under 808 nm NIR irradiation (1 W cm− 2) within 5 min at the bottom temperature of 32 ℃, which was changed positively correlated with DA concentration, and the near-infrared image showed that the temperature in the middle position was highest (white color). Moreover, 0.5 DA, 1 DA, and 1.5 DA-Yang hydrogel persistent overheating after 10 min due to evaporation of water. Unlikely, Yin hydrogel as the middle part of Tai-Chi hydrogel decreased the temperature of the surrounding Yang hydrogel under the same condition (Fig. 3a), which suggested that Yin hydrogel would prevent the wound from overheating and damage during the photothermal therapy. In addition, compared with pure Yang hydrogel, Yin hydrogel improved the temperature stability of Tai-Chi hydrogel from 5 min to 20 min, which demonstrated that Yin hydrogel have the potential to sustain the temperature stable during photothermal therapy (Fig. 3a, b, Fig. S6). Different with 1 DA- Tai-Chi and 1.5 DA-Tai-Chi hydrogel groups, the 0.5 DA-Tai-Chi hydrogel could maintain the temperature from 41.13 ± 0.31 ℃ at 5 min to 41.17 ± 0.23 ℃ at 20 min under 808 nm NIR irradiation (Fig. 3b), that indicated 0.5 DA-Tai-Chi hydrogel met the requirement of following experiment. Skin temperature varies greatly in different parts and individuals, we detected the temperature of the back skin of rat before in vivo experiment, the minimum temperature and maximum temperature were 32.2 ℃ and 34.5 ℃, with the average temperature was 34 ℃ measured by FLIR Tools software. Therefore, the photothermal property of 0.5 DA-Tai-Chi hydrogel at different temperature bottom plate were measured. As shown in Fig. 3c, the bottom temperature had a greatly influence on the temperature of the hydrogel under 808 nm NIR irradiation (1 W cm− 2). The maximum temperature of 0.5 DA-Tai-Chi hydrogel were 41.5 ℃, 42.2 ℃, 42.6 ℃ and 42.5 ℃ under 808 nm NIR irradiation (1 W cm− 2) at 5, 10, 15, and 20 min, respectively, which was slightly higher than the experimental temperature by 40–41℃ [23, 26]. To explore the suitable experimental condition, 0.5 DA-Tai-Chi hydrogel were irradiated by NIR laser with different power density (0.8, 0.9, 1 W cm− 2). The result showed that the maximum temperature of the Tai-Chi hydrogel was 38.8 ℃, 40.1 ℃ and 41.5 ℃ at 5 min, respectively. The maximum temperature was 39.4 ℃, 40.9 ℃, and 42.2 ℃ at 10 min, respectively (Fig. 3d), which indicated 0.9 W cm− 2 was selected as the following in vitro and in vivo experiment condition. The photothermal stability of 0.5 DA-Tai-Chi hydrogel was also confirmed by the temperature measurement with three laser irradiation cycles (Fig. 3e). To explore the suitable photothermal condition for cell experiments, 0.5 DA-Yang hydrogel was attached to the bottom of a 24-well plate filled with medium and was irradiated by laser with different power density (0.67, 0.78 W cm− 2) at room temperature, the change of maximum and average temperature was recorded in Fig. S7. The maximum and average temperature were 41 ℃ and 40 ℃ at 5 min under power density of 0.78 W cm− 2, which was suitable for cell experiments. In addition, to evaluate the possibility of Tai-Chi hydrogel as a wound dressing on human skin, we conducted the photothermal experiment of Tai-Chi hydrogel on human skin (1W cm− 2, 20 min). As shown in Fig. S8, it could be clearly seen that the temperature difference between the Yin hydrogel and the Yang hydrogel on the upper surface of Tai-Chi hydrogel, which exhibited same result as in vitro simulation before. Interestingly, the hydrogel removed after 20 min irradiation, thermal images presented that the skin temperature under Yin hydrogel of 0 DA-Tai-Chi hydrogel was higher than under Yang hydrogel, while the skin under Yin hydrogel of 0.5 DA-Tai-Chi and 1 DA-Tai-Chi hydrogel presented that mild warm diffused from Yang hydrogel. This result indicated that Yin hydrogel has the ability to adjust the wound area temperature according to the environment, which prevented overheating of central part and maintained the mild heat of the wound under NIR irradiation.
3.3 In Vitro Macrophages Polarization
To simulate the process of Yang part in Tai-Chi hydrogel treated with heat stimulation caused by NIR irradiation in vitro on wound healing, a model was designed as shown in Fig. 4a, which was performed to study the proliferation and differentiation of macrophages.
As shown in Fig. 4b, RAW 264.7 macrophage in Yang-NIR group displayed M2 phenotype compared with the Control and Yang groups (M0 macrophages) according to the staining of actin filaments labeled with FITC-phalloidin, while the relative cell proliferation in RAW 264.7 macrophages exhibited no obvious difference among three groups (Fig. 4c). The degree of cell elongation, which was defined as long axis length to short axis length, exhibited a significant higher ratio in Yang-NIR group than other two groups at day 3 (Fig. 4e).
To further confirm macrophages polarization status, immunofluorescence staining of CD206 (a surface marker of M2 macrophage) was performed in RAW 264.7 macrophages among three groups. Remarkably higher level of CD206 positive staining could be observed in Yang-NIR than other two groups at day 3 (Fig. S9a), which was consistent with statistical data of fluorescence intensity (Fig. S9b). Furthermore, the polarization status of M2 macrophages in Yang-NIR group exhibited more obviously at day 5, which was specifically manifested by more elongated and stretched cell morphology and larger nuclei (Fig. 4d), and the fluorescence intensity was extremely significantly higher than that two groups (Fig. 4f).
3.4 Application of Tai-Chi Hydrogel in Diabetic Wound Healing
Above experiments confirmed that photothermal properties of Yang hydrogel combined with NIR irradiation could promote the proliferation and migration of fibroblasts and vascular endothelial cells in vitro. Cutaneous full-thickness wounds were created on the back of diabetic rats to verify whether Tai-Chi hydrogel could accelerate wound healing in vivo. Fig. S10a showed the treatment of Tai-Chi hydrogel combined with NIR irradiation on wounds, and Fig. S10b presented the whole experimental process. Interestingly, compared with the relative wound area of the Control group (71.39 ± 6.90%), Tai-Chi-NIR group (37.44 ± 6.77%) was significantly reduced after 3 days, and the Tai-Chi group individually (53.86 ± 5.92%) also had promoting effect. After 7 days, the relative wound area in Tai-Chi-NIR group was reduced up to 16.65 ± 4.69%, followed by Tai-Chi group (25.17 ± 3.18%) and Control group (35.35 ± 6.74%). Subsequently, there was no significant difference between day 10 (11.52 ± 2.00%) and day 14 (11.97 ± 2.41%) in the Tai-Chi-NIR group, while the area in the Tai-Chi group was still shrinking compared with the Control group on day 14 (Fig. 5a, b, c). The result indicated that wounds in Tai-Chi-NIR group were completely closed first, and Tai-Chi hydrogel combined with NIR irradiation had obviously promoting effect on wound healing in the early stage (day 3).
H&E staining was used to evaluate the wound status and tissue regeneration at day 3, 7 and 14, respectively. As shown in Fig. 5d, with the progress of wound healing, granulation tissues in wound beds were experienced a process of thickening first and then thinning. Figure 5g presented statistical data results of granulation tissue thickness in each group at day 3,7 and 14. The thickness of granulation tissues that contained a lot of blood vessels and fibroblasts in Tai-Chi-NIR groups (1.09 ± 0.22 mm) were significantly thicker than Tai-Chi groups (0.62 ± 0.12 mm) and Control groups (0.37 ± 0.12 mm) after 3 days, and the direction of granulation tissue formation was from bottom to top, from the edge to the center (Fig. 5d, g). This directionality may be due to the photothermal characteristics of Tai-Chi hydrogel, with normal tissue around heating and mild heat of wound beds keeping, to promote the migration of fibroblasts. Compared with the Control group, hair follicles (red arrows) and sebaceous glands (yellow arrows) were appeared in the wound bed of Tai-Chi group after 14 days, but the wound morphology were different with the surrounding normal tissue. However, in the Tai-Chi-NIR group, except for a few tissues adjacent to epidermis, tissues with regenerated mature hair follicles and sebaceous glands in wound bed had no difference with normal tissue, which indicated that wounds in Tai-Chi-NIR groups entered tissue remolding phase earlier.
Re-epithelialization is an important index in the process of wound repair, which could prevent wound tissues been exposed to the external environment. Although epidermis layer (green arrows) could be roughly observed from H&E staining at day 7 and day 14, the process of epidermal regeneration cannot be well presented in the early stage of wound healing (Fig. 5c). Cytokeratin 14 (a epidermal cell marker, CK-14) antibody and epidermal growth factor (EGF) antibody were used to label epidermal cells in granulation tissue [27, 28]. It could be clearly observed that more CK14 and EGF positive staining in the Tai-Chi-NIR group after 3 days, followed by the Tai-Chi group and the Control group, which indicated that the re-epithelialization degree of wounds in the Tai-Chi-NIR group was higher than other two groups. (Fig. 5e, f). Almost complete epidermal layer marked by CK14 were formed in the Tai-Chi group and Tai-Chi-NIR group after 7 days, while the Control group still incompletely (Fig. S11). Keratinocyte growth factor (KGF), also named FGF 7 [29], is produced by fibroblasts [30]. Increased expression of KGF during re-epithelialization could induce the proliferation and migration of keratinocytes [27, 30–32]. RT-qPCR was used to monitor the expression level of KGF and EGF of wounds in each group at day 3, 7 and 14, respectively (Fig. 5h, i). Compared with the Control group and the Tai-Chi group, the expression level of KGF and EGF in the Tai-Chi-NIR group was significantly increased at day 3, which corresponded to results before.
Insulin-like growth factor 1 (IGF-1) is a polypeptide protein with similar molecular structure to insulin, which could reduce blood glucose and has a good therapeutic effect on type II diabetes [33, 34]. Recent studies have shown that increasing IGF-1 level could accelerate healing of diabetic wounds by promoting vascularization [35–37], which is associated with activation of the PI3K / Akt signaling pathway when IGF-1 binds to IGF-1R [38], Akt1 mediates angiogenesis by phosphorylating multiple angiogenic substrates [39]. We detected the expression of IGF-1 in the whole wound healing process by RT-qPCR. As shown in Fig. 5j, the expression level of IGF-1 in the Tai-Chi-NIR group was significantly higher than in the Control group and Tai-Chi hydrogel group at day 3 and 7, while the expression level of IGF-1 down regulated after 14 days with no significantly difference to other groups, which demonstrated that warm stimulation caused by Tai-Chi hydrogel under NIR irradiation improved the expression level of IGF-1 in wound tissue in proliferation phase, but have no risk of causing potential adverse effects due to no persistent highly expression of IGF-1 [40]. Furthermore, IGF-1 positive staining in granulation tissue of Tai-Chi-NIR group was significantly higher than that of other two groups (the white line was the boundary of IGF-1 content in granulation tissue) (Fig. S12). Combined to the result of H&E staining and RT-qPCR, IGF-1 expression in granulation tissue was positively correlated with the degree of tissue repair. (Fig. 5a, d, j, Fig. S12).
To sum up, results above suggested that wounds treated with Tai-Chi hydrogel and NIR irradiation together significantly accelerated wound closure and tissue remolding, especially in the early stage (day 3, inflammatory phase).
3.5 Inflammation Regulation and M2 Polarization of Macrophages In Vivo
Diabetic wound is one kind of chronic wound with long inflammatory period, timely shifting the immune microenvironment from pro-inflammatory to anti-inflammatory is importantly, which could facilitate migration and proliferation of fibroblasts and promote vascularization and nutrition supply [41]. Macrophages exhibit different phenotypes and functional performance, which is closely related to their surrounding immune microenvironment. Macrophages are mainly classified into two main subsets: 1) M1 macrophages, which could produce high levels of proinflammatory cytokines, including IL-1β, IL-6 [42] and TNF-α, to facilitate leukocyte recruitment and activation been injured; 2) M2 macrophages, which participate in wound repair and tissue remodeling [43], and participate in secretion of anti-inflammatory cytokine (IL-10) to reduce the inflammatory response [44–46]. Recent studies have shown that wound healing process can be accelerated by promoting macrophages convert to M2 phenotype, especially in diabetic wound [41, 47–51].
In vitro experiment proved that Yang hydrogel cooperated with NIR irradiation could stimulate macrophages polarization to M2 phenotype, but the effect of Tai-Chi hydrogel and in combination with NIR irradiation in vivo still underdetermined. To monitor the M2 phenotype macrophages status during the whole wound repair process, CD206 antibodies were labeled on M2 macrophages in wounds. Compared with the Control group, the Tai-Chi group showed the performance of promoting macrophages to M2 polarization at day 3, which in combination with NIR irradiation significantly increased the number of M2 phenotype macrophages in the Tai-Chi-NIR group. At day 7, the number of M2 macrophages in the Control and Tai-Chi group continuous increasing, while the number of M2 macrophages in Tai-Chi-NIR group was similar with that at day 3 (Fig. 6a, c). During the wound healing process, M2 macrophages increased first and then decreased as time goes on, followed disappeared with wound repair completely (Fig. 6a), which exhibited the same tendency consistent with the research before [51].
Results presented before determined that Yang hydrogel could not stimulate macrophages polarization in vitro, however, more CD206 positive staining could be observed in Tai-Chi group than the Control group, which speculated that treated with Tai-Chi hydrogel itself also has effect on the microenvironment of diabetic wounds.
Three proinflammatory factors (IL-1β, IL-6, TNF-α) and two anti-inflammatory cytokines (IL-10, IL-22) were performed to investigate the effect of Tai-Chi hydrogel and synergistic effect with NIR irradiation on the wound microenvironment of macrophages. As shown in Fig. 6b (i), CD68-labeled macrophages exhibited much lower levels of IL-1β positive staining in Tai-Chi-NIR group than the Control and Tai-Chi group at day 3, which exhibited the same result at day 7 (Fig. S13a). Relative gene expression of IL-1β also demonstrated that mRNA level of IL-1β in Tai-Chi-NIR was significantly lower than other two groups at day 3 and 7 (Fig. 6d). The percentage of macrophages expressed IL-1β in Tai-Chi-NIR group was remarkably lower than the Control and Tai-Chi group (Fig. 6f). The result demonstrated that the level reduction of IL-1β in the wound was related to the photothermal therapy but not Tai-Chi hydrogel itself, which indicated that Yang hydrogel played the main role in regulating level of IL-1β in wound immune microenvironment.
Interestingly, the level of IL-6 positive staining in Tai-Chi and Tai-Chi-NIR groups have no significant difference at day 3, but these two groups were significantly lower than the Control group represented in Fig. 6b (ii). The mRNA level of IL-6 in Tai-Chi and Tai-Chi-NIR groups were significantly lower than the control group at day 3, 7 and 14 (Fig. 6e) demonstrated the same result, which suggested that Tai-Chi hydrogel individually could decrease the expression level of IL-6 and NIR irradiation synergistic therapy has no obvious effect on IL-6 expression. During the early stage of wound healing process, we found a phenomenon that wounds in Tai-Chi and Tai-Chi-NIR group were cleaning and drying when changing dressing every time, while wounds in the Control group accumulated a lot of pus (Fig. S14). This effect indicated that Tai-Chi hydrogel has a potential ability to reduce the risk of wound sepsis as a wound dressing, which might be attributed to Yin hydrogel (BC) could absorb pus well due to its good swelling performance of natural bacterial cellulose as inner part of Tai-Chi hydrogel [11, 14, 52], while sepsis was positively correlated with IL-6 expression [53–55]. Above result demonstrated that the level reduction of IL-6 in the wound was related to the Tai-Chi hydrogel itself but not photothermal therapy, which indicated that Yin hydrogel played the main role in regulating level of IL-6 in wound microenvironment.
However, the result of immunohistochemistry staining and RT-qPCR of TNF-α showed that there was no significant difference among three groups, which indicated that the promotion effect of wound healing in this study have no relationship with TNF-α (Fig. S15).
The polarization of macrophages to M2 phenotype was accompanied by higher expression of IL-10 [44]. More IL-10 positive staining could be observed in Tai-Chi-NIR group at day 3, followed by the Tai-Chi group and the Control group, respectively (Fig. 6b (iii)), quantitative data of IL-10 positive coverage per field was shown in Fig. 6g. In addition, there were still have some IL-10 positive staining could be observed around CD68-labeled macrophages in Tai-Chi-NIR group at day 7, while the Control and Tai-Chi groups were not (Fig. S13b), the RT-qPCR result demonstrated the expression of IL-10 mRNA level was higher at day 3 than day 7 and 14 (Fig. S13c), that meaned more IL-10 in the Tai-Chi-NIR group. As a member of IL-10 family, IL-22 has the effect of promoting wound healing and tissue regeneration [56], which presented superior efficacy treatment than FDGF or VEGF treatments in infection diabetic wound [57, 58]. In this study, IL-22 showed a higher expression level tendency by RT-qPCR in Tai-Chi-NIR group at day 3 and 7, followed by Tai-Chi group and the Control group (Fig. S13d).
In conclusion, Tai-Chi hydrogel has the ability to regulate diabetic wound inflammatory microenvironment. Yin hydrogel could reduce the level of IL-6 and increase the level of IL-10, thereby promoting the polarization of macrophages to M2 phenotype. Under NIR irradiation, warm stimulation produced by Yang hydrogel could rapidly reduce IL-1β level and further increase IL-10 level, which further promoted macrophages from M1 to M2 phenotype, to accelerate wound enter proliferation phase [41, 59, 60].
3.6 Collagen Synthesis In Vivo
Collagen deposition is an important index to evaluate wound healing and tissue remolding, which is synthesized and secreted by fibroblasts proliferation and migration. Figure 7a, e exhibited the representative Masson’s trichromatic staining images and statistical data of collagen deposition in each group on day 3, 7 and 14, respectively. Collagen deposition in Tai-Chi-NIR group (11.78 ± 2.31%) displayed significantly higher than in Tai-Chi group (9.18 ± 1.20%) and the Control group (8.69 ± 2.85%) after 3 days, and the direction of collagen deposition in Tai-Chi-NIR group is from bottom to top, from edge to center (Fig. 7a, e). An obviously boundary line about collagen density, which revealed that more collagen in the lower part than that in the upper part of granulation tissue, could be observed in Tai-Chi-NIR group at day 7 (Fig. 7a), collagen deposition in Tai-Chi-NIR group (37.28 ± 7.59%) was further remarkable higher than in Tai-Chi group (17.30 ± 2.30%) and the Control group (15.62 ± 2.83%). While on the 14th day, the collagen deposition in the Control group, Tai-Chi group and Tai-Chi-NIR group was 40.76 ± 4.36%, 51.09 ± 3.99% and 66.22 ± 8.25%, respectively, Tai-Chi-NIR group was still significantly higher than other two groups (Fig. 6e). In addition, except a small portion of collagen staining in wound area near to the epidermis was similar with the Tai-Chi group and the Control group, the staining of collagen in other parts of wound had no significant difference with the surrounding normal tissues at day 14, this trend was consistent with H&E staining results.
SA100A4, is also known as FSP1 (Fibroblast-specific protein 1), which is a fibroblast marker in remodeling tissues of different organs. S100A4+ cells are mainly fibroblasts of the skin [61], kidneys [62], lungs [63] and normal heart [64, 65]. As shown in Fig. 7b, c, more PCNA (proliferating cell nuclear antigen) and S100A4 positive cells in Tai-Chi-NIR group could be observed at day 3, followed by the Tai-Chi group and the Control group, which indicated that the number of fibroblasts in proliferation phase was largest in the Tai-Chi-NIR group. The positive staining of COL I increased from day 3 to day 7 in all groups, but more COL I positive staining could be observed in the Tai-Chi-NIR group, followed by the Tai-Chi group and the Control group (Fig. 7d, g). However, COL I positive staining in the Tai-Chi-NIR group rapidly decreased at day 14, followed by the Tai-Chi group, while the the Control group still increased (Fig. 7g, Fig. S16). Interestingly, COL I positive coverage in the Tai-Chi-NIR group (28.61 ± 2.25%) at day 7 reached the same level as the Control group (29.23 ± 3.04%) at day 14 (Fig. 7g). The results of Masson’s trichromatic staining and immunostaining of COL I suggested that wounds in the Tai-Chi-NIR group entered the tissue remodeling stage ahead of time due to type I collagen in the granulation tissue will be largely replaced by type III collagen to withstand stronger mechanical stress during the tissue remodeling stage of wound healing [66].
Fibroblast activation protein α (FAPα) is an antigen substance expressed on the surface of activated fibroblasts in granulation tissue during wound healing and tumor stroma [67–70]. Figure 7f showed that the relative gene expression of FAPα in the Tai-Chi-NIR group was highest in three groups on day 3, followed by the Tai-Chi group and the Control group. Subsequently, compared with the Control group, the relative gene expression of FAPα decreased in the Tai-Chi group and the Tai-Chi-NIR group after 7 days and 14 days. These results above indicated that Tai-Chi hydrogel could promote fibroblasts activation in the early stage of wound healing (day 3), and this activation effect could be further enhanced in cooperation with NIR irradiation, and this activation effect was orderly that disappeared with the process of wound repair.
In a word, Tai-Chi hydrogel combined with NIR irradiation could promote fibroblasts proliferation and collagen deposition in diabetes wounds, especially in the early stage of wound healing. In addition, this therapy method will not lead to excessive proliferation of fibroblasts and excessive deposition of type I collagen, resulting in pathological scar hyperplasia.
3.7 Neovascularization In Vivo
Angiogenesis affects the process of wound healing, especially in chronic wounds. Yang hydrogel with NIR irradiation have been confirmed to promote proliferation and migration of HUVEC cells in vitro, but the effect in vivo still undetermined. Next, immunohistochemical staining of CD34 (vascular endothelial cell marker) was used to evaluate the formation of new blood vessels during wound healing process. In the early stage of wound repairing (day 3), the positive coverage of CD34 staining in granulation tissue of Tai-Chi-NIR group was significantly higher than other two groups, indicated that neovascularization in the wound was significantly enhanced (Fig. 8a, b). Moreover, the positive area rate of CD34 staining in Tai-Chi-NIR group was significantly lower than other two groups at day 14, which was consistent with the phenomenon of vascular reduction in the later stage of wound healing. In addition, the diameter of blood vessel in Tai-Chi-NIR group was significantly smaller than other two groups at day 7 and day 14 (Fig. 8a), and the blood vessel diameter in Tai-Chi group was also smaller than control group. To monitor the dynamic process of angiogenesis during wound healing, relative gene expression of VEGF and α-SMA in granulation tissue were detected by RT-qPCR. Results in Fig. 8c, d demonstrated that the expression of two genes related to angiogenesis at day 14 was lower than day 3 and day 7, which was consistent with the immunohistochemical results in Fig. 8a.
Furthermore, newly blood vessels marked with α-SMA (vascular smooth muscle cell marker) were observed and measured by immunofluorescence staining at day 3 (Fig. 8e, f). Wounds treated with Tai-Chi hydrogel and Tai-Chi-NIR exhibited high level of α-SMA staining than Control group, especially Tai-Chi hydrogel combined with NIR treatment further increased positive-staining of α-SMA than Tai-Chi hydrogel individually, which was consistent with the RT-qPCR result of a-SMA at day 3 (Fig. 8d, e, f). Combined with the fluorescence staining result of α-SMA (Fig. 8e, f) and the statistical result of CD34 (Fig. 8b), which indicated that the promotion of angiogenesis by Tai-Chi hydrogel individually was related to its promotion of vascular smooth muscle cells but not vascular endothelial cells. Moreover, the synergistic effect of Tai-Chi hydrogel with NIR could promoted the proliferation of vascular endothelial cells and further promotion of vascular smooth muscle cells (Fig. 8e, f). In addition, the relative gene expression of VEGF in wounds demonstrated no significant difference among three groups might be due to vascular endothelial cells were monolayer cells in newly blood vessels that resulting in less content in the regenerated tissue to be detected (Fig. 8c) [71]. Therefore, all results above illustrated the accelerated effect of Tai-Chi hydrogel individually or in combination with NIR irradiation on angiogenesis during diabetic wound healing process.
As mentioned above, compared with the Control group, higher expression of angiogenesis-related indexes (α-SMA, IGF-1) and fibroblast-related indexes (PCNA, S100A4 and FAPα) in the wounds of Tai-Chi group might be related to the improvement of the wound inflammatory microenvironment by Yin hydrogel, which suggested that Tai-Chi hydrogel could be a potential dressing on chronic wound healing.
3.8 Effects of Heat Shock Proteins In Vivo
Decreased the level of heat shock protein (hsp) may prolong the healing process of diabetic wound [72, 73], mild heat stimulation could increase the level of hsp70 and hsp90 in wound tissues and accelerate the process wound healing [23, 74]. As shown in Fig. S17a, compared with the Control group and Tai-Chi group, relative expression of hsp70 in Tai-Chi-NIR group was significantly increased and consistent with the wound repair result at day 3, which suggested that increased hsp70 was also play an important role in accelerating wound repair at day 3, due to reason that higher level of hsp70 could promote fibroblasts migration [73]. Different with previous study, the expression level of hsp90α in Tai-Chi-NIR group has no significant difference with other two groups (Fig. S17b), which may be related to that hsp70 is more sensitive to heat stimulation.