Thermogels for Cutaneous Tumor

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Defective Black Nano-Titania/Thermogels for Cutaneous Tumor-Induced Therapy and Healing Xiaocheng Wang, Bing Ma, Jianmin Xue, Jinfu Wu, Jiang Chang, and Chengtie Wu Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00367 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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Defective

Black

Nano-Titania/Thermogels

for

Cutaneous Tumor-Induced Therapy and Healing Xiaocheng Wang1, 2, Bing Ma1, Jianmin Xue1, 2, JinFu Wu1, 2, Jiang Chang1, Chengtie Wu1, 2* 1. State Key Laboratory of High Performance Ceramics and Superfine Microstructure, Shanghai Institute of Ceramics, Chinese Academy of Sciences, 1295 Dingxi Road, Shanghai 200050, People’s Republic of China. 2. Center of Materials Science and Optoelectronics Engineering, University of Chinese Academy of Sciences, 19 Yuquan Road, Beijing 100049, People’s Republic of China.

* Corresponding author: Chengtie Wu E-mail: [email protected] (C. Wu); Tel: +86-21-52412249.

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ABSTRACT Current challenges in cutaneous tumor therapy are healing the skin wounds resulted from surgical resection and eliminating possible residual tumor cells to prevent recurrence. To address this issue, bifunctional biomaterials equipped with effective tumor therapeutic capacity for skin cancers and simultaneous tissue regenerative ability for wound closure are highly recommended. Herein, we report an injectable thermosensitive hydrogel (named as BT-CTS thermogel) by integration of nano-sized black titania (B-TiO2-x, ~ 50 nm) nanoparticles into a chitosan (CTS) matrix. The BTiO2-x nanocrystal exhibits a crystalline/amorphous core-shell structure with abundant oxygen vacancies, which endows the BT-CTS thermogels with simultaneous photothermal therapy (PTT) and photodynamic therapy (PDT) effects under singlewavelength near infrared (NIR) laser irradiation, leading to excellent therapeutic effect on skin tumors in vitro and in vivo. Moreover, the BT-CTS thermogel not only supports the adhesion, proliferation, migration of normal skin cells, but also facilitates skin tissue regeneration in a murine chronic wound model. Therefore, such BT-CTS thermogel with easy injectability, excellent thermostability and simultaneous PTT/PDT efficacy as well as tissue regenerative activity offers a promising pathway for healing cutaneous tumor-induced wounds. KEYWORDS: Black titania, oxygen vacancies, thermogel, tumor therapy, wound healing

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Surgical excision remains the mainstay for the treatment of cutaneous cancers nowadays.1,2 Unfortunately, it is extremely difficult to completely eradicate tumor cells and restore normal skin function after the resection of large cancerous tissues.3,4 Therefore, appropriate treatments for skin cancers after surgery should emphasize both tissue reconstruction and effective inhibition of tumor reoccurrence. Our recent studies have developed a kind of bifunctional electrospun membranes by incorporating the photothermal agents (e.g., cuprous sulfide nanoflowers5 and copper silicate hollow microspheres6) into electrospun fibers. Although the preliminary results have confirmed the hypothesis of combining skin tumor therapy with skin wound healing by using a bifunctional biomaterial is highly possible, such bifunctional electrospun membranes are still far from satisfying considering their insufficient mechanical stability, difficult manipulation and inhomogeneous distribution of photothermal particles. Therefore, it is imperative to develop an innovative strategy for repairing the cutaneous tumor-induced defects after routine surgical excision of large tumor tissues. Apart from photothermal therapy (PTT), photodynamic therapy (PDT) has also been considered as a non-invasive modality for treating various cancers in the past decades.79

Both PTT and PDT involve the administration of photosensitizing agents with high

optical absorbance, to effectively transfer photo energy into regional hyperthermia or generate cytotoxic reactive oxygen species (ROS) at the tumor site respectively, leading to irreversible damage to the tumor cells.10-13 As compared to either PDT or PTT alone, the combination of the two modalities has been demonstrated to greatly improve therapeutic effect on cancer cells.14-18 However, current combined strategies mostly 3


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utilize two different functional components and/or separated laser sources to trigger PDT and PTT respectively, resulting in a complicated and prolonged treatment.19 Hence, it would be of great significance to achieve simultaneous and synergistic PDT/PTT under a single laser irradiation. Recently, black titania (B-TiO2-x) nanoparticles with oxygen-deficient structures have been employed as photosensitizers for tumor therapy under the single-wavelength NIR irradiation.11,20 However, the nanoparticles still remain great challenges when directly applied for healing large-sized wounds, such as limited bioactivity, inevitable particle aggregation, rapid leakage, inhomogeneous distribution, acute wound inflammation at the wound sites. These issues could be addressed by incorporation of the nanoparticles into a proper wound dressing biomaterial to satisfy the dual demands for locally treating tumor cells and reconstructing resected skin tissues. Injectable hydrogels have emerged as promising wound dressing materials because they can fill irregular defects, provide a moist wound environment, serve as a barrier to microorganisms and delivery therapeutic agents to the injury sites.21-24 Particularly, chitosan (CTS)-based hydrogels have been highly pursued in wound healing applications thanks to their intrinsic biocompatibility, biodegradability, hemostatic property, antibacterial activity and stimulation of wound healing.25-27 A series of CTS thermogels developed by Lerouge et al. has exhibited very attractive features including easy injectability at room temperature, rapid gelation and high compression strength at physiological temperature, excellent compatibility for cell encapsulation and acceptable biodegradation, when the gels were prepared in the presence of phosphate 4


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buffer and sodium hydrogen carbonate (NaHCO3).28,29 Therefore, it is highly inspired to realize tumor therapy and wound healing within a single platform via incorporation of B-TiO2-x nanoparticles into the CTS thermogels. It is expected that the B-TiO2-x nanoparticles incorporated CTS thermogels could not only act as a localized therapeutic agent for clearing the residual tumor cells under the NIR irradiation in a short time, but also play an active role in stimulating the long-term tissue regeneration at the defect sites after resection of skin tumors. To the best of our knowledge, the concept and design strategy for such an intelligent bifunctional platform have not yet been reported so far. Herein, we report an injectable bifunctional thermosensitive hydrogel (named as BTCTS thermogel) by incorporating Mg-containing B-TiO2-x nanoparticles into the CTS networks for localized tumor therapy and simultaneous skin wound healing. The BTiO2-x nanoparticles were firstly synthesized via a facile Mg-thermic reduction reaction and then embedded into the CTS networks, rendering the composite thermogel synergistic PTT/PDT performance under the single-wavelength NIR irradiation (808 nm). The morphology, chemical composition, PTT/PDT effect, thermostability, and rheological property of BT-CTS thermogels were systematically characterized. Subsequently, the anticancer efficacy was investigated to kill skin tumor cells in vitro and inhibit the skin tumor growth in vivo. Finally, the intrinsic tissue regenerative ability of BT-CTS thermogels was evaluated by healing both skin tumor-induced wounds and full-thickness chronic wounds. In our study, Mg-thermic reduction was utilized to create structure defects in TiO2 5


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nanocrystals as previously reported.30 TiO2 and Mg powders (molar ratio of 2:1) were mixed, ground up and calcined at 650 °C under Ar atmosphere for 4 h. The Mg-reduced TiO2 nanoparticles were obtained after washing with excess HCl to remove the residual elemental Mg. Their morphology and structure (Figure 1a-f) were examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). As compared to the white pristine TiO2 (25 nm), the obtained nanoparticles appeared black with slightly growth of the grains (~ 50 nm) after thermal treatment at 650 °C (Figure 1a,d and Figure S1). The black color of Mg-reduced TiO2 samples could be derived from the enhanced absorption of visible light, and thus we named them as B-TiO2-x. The white TiO2 was highly crystalline with well-resolved lattice fringes even at the nanocrystal surface (Figure 1b and Figure S1 top), in dramatically contrast to a crystalline/amorphous core-shell structure of B-TiO2-x nanocrystal (Figure 1e and Figure S1 bottom). The core displayed a well-defined (101) lattice plane with a typical anatase plane distance of 0.35 nm (Figure 1b,e), while the distance between adjacent lattice planes was highly-distorted at the edge of the B-TiO2-x nanocrystal. Geometric phase analysis (GPA, Figure 1c,f) was applied to detect the nanoscale lattice distortion induced by crystal defects via quantitatively measuring the local strains as revealed in the high-resolution TEM (HRTEM) observation.31-33 The strain fields were represented by strain tensor components εxx, εyy, εxy and γxy with a scale range of -0.5 to 0.5. The positive value represented compressive strain while the negative value represented tensile strain. The results were interpreted as false-color maps where regions in accord with a given periodicity were presented with the same color. It was found that the shear 6


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strain in the pristine TiO2 and the interior of B-TiO2-x approximated to zero, suggesting a perfect non-sheared state and few atomic displacements in the crystal lattices. However, many color discontinuous points corresponding to the dislocation core positions were presented in the phase images of B-TiO2-x (marked by white arrows in Figure 1f), indicating high density of atom defects. Moreover, X-ray diffraction (XRD) patterns (Figure 1g) showed the crystalline structure with the anatase phase (PDF # 211272) in majority, as well as Ti4O7 (2TiO2∙Ti2O3) and Mg2TiO4 (2MgO∙TiO2) in minority for B-TiO2-x, indicating the introduction of Ti3+ and Mg2+ into TiO2 nanocrystals possibly through the reduction reaction of 2TiO2 + Mg → Ti2O3 + MgO, which was consistent with the results of X-ray photoelectron spectroscopy (XPS, Figure 1h and Figure S2). The characteristic peak of Ti3+ shown in the Ti 2p XPS spectra could be ascribed to the generation of oxygen vacancies, which produced excess electrons and thereby transformed Ti4+ into Ti3+.34,35 In addition, Raman spectra were examined to investigate the surface chemical compositions and corresponding oxidation states. As depicted in Figure 1i, both TiO2 and B-TiO2-x showed five characteristic peaks of anatase. Notably, the blue shift and broadening of the peak at ~ 145 cm-1 for the B-TiO2-x further verified the existence of a considerable number of oxygen vacancies after Mg reduction treatment.35,36 Due to the presence of oxygen vacancies and disorder-induced lattice strain within BTiO2-x nanocrystals, the light absorption of B-TiO2-x could be effectively expanded to the visible and NIR regions (Figure S3a).30,34,37,38 The optical absorption intensity at 808 nm, a typical wavelength used for photothermal therapy, linearly increased with 7


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the concentrations of B-TiO2-x (Figure S3b). The real-time temperature elevation of different B-TiO2-x aqueous dispersions (0 ~ 800 ppm, 500 μL) was then recorded under the 808-nm irradiation for 5 min. It was found that the B-TiO2-x aqueous dispersions showed sharp temperature increase with strong dependence on concentrations, in marked contrast to a mild temperature elevation of pure water (Figure S4). In addition, XRD analysis has been conducted to investigate the phase change of black titania particles after NIR irradiation. The results (Figure S5) showed no obvious difference between the XRD patterns before and after photothermal treatment (808 nm, 0.48 W/cm2, 30 min). Pristine TiO2 has been previously utilized as a photocatalyst for killing cancer cells upon ultraviolet (UV) light irradiation, because of the photoinduced generation of various ROS including singlet oxygen (1O2), hydroxyl radicals (∙OH) and superoxide anion (∙O2-).39-41 It is interesting that the B-TiO2-x nanoparticles exhibited the capability to generate 1O2 when they were exposed to the 808-nm irradiation. For 1O

2

detection, 1,3-diphenylisobenzofuran (DPBF) was utilized as a chemical probe

because it could be oxidative degraded in the presence of 1O2 with gradual decline of its absorption at 410 nm.20, 42 Our results revealed an obvious decrease in absorption intensity of DPBF solution containing B-TiO2-x under NIR irradiation (Figure S6a), while the white TiO2 group with NIR laser alone showed a slight decrement (Figure S6b), indicating the unique NIR-induced 1O2 generation capacity of B-TiO2-x. It is believed that the bandgap narrowing derived from the oxygen vacancies in the disordered surface layers could be responsible for the “red shift” of the optical absorption spectra from UV to NIR region.34,35,43

Thus, the B-TiO2-x nanoparticle was

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expected to serve as an excellent NIR-triggered synergistic PTT/PDT agent for tumor therapy. For repairing the tumor-induced cutaneous defects, B-TiO2-x powders failed to provide sufficient mechanical strength and porous structure for inducing tissue ingrowth when directly applied in the skin wounds. Therefore, a series of BT-CTS thermogels was prepared by incorporating different amounts of B-TiO2-x nanoparticles into a CTS thermogel.28 The aqueous BT-CTS blend was firstly prepared by directly dispersing a given mass of B-TiO2-x nanoparticles (0, 5, 10, 20 and 40 mg) in the acidic CTS solution (6 mL, 3.33 w/v % in 0.1 M HCl). The gelling agent was prepared by dissolving NaHCO3 (0.1875 mol) in the phosphate buffer (0.1 M, 1000 mL) at pH = 8. When the BT-CTS blend was mixed with the gelling agent (volume ratio of 3:2, Figure 2a) by two connected syringes and then kept at physiological temperature (37 ℃), the mixture immediately lost its fluidity and turned into a stagnant hydrogel (Figure 2b). All thermogels were fabricated to reach a final CTS concentration of 2 % (w/v) and BTiO2-x contents of 0, 0.5, 1, 2, 4 ‰ (w/v), and thus denoted as 0T-, 0.5BT-, 1BT-, 2BT-, and 4BT-CTS thermogels, respectively. White thermogels named as 0.5WT-, 1WT-, 2WT, and 4WT-CTS thermogels were prepared accordingly by incorporating pristine TiO2 nanoparticles into the CTS matrix. The color of BT-CTS thermogels changed gradually from grey to dark black as the dosage of B-TiO2-x nanoparticles increased (Figure 2b and Figure S7). SEM analysis of the freeze-dried thermogels showed their highly-porous

microstructure

(100-300

μm)

with

inorganic

nanoparticles

homogenously embedded in the biopolymer matrix and B-TiO2-x contents within the 9


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gels increased (Figure 2c and Figure S8). Elements Ti and Mg (atom ratio of 6.14:1) were successfully imported into the BT-CTS systems, as demonstrated by energydispersive spectroscopy (EDS) elemental mapping (Figure 2d-h, Figure S9 and S10). When the gels (1 mL) were immersed in the aqueous Tris-HCl buffer solution (pH~7.4, 10 mL) at 37 ℃, Mg ions could be sustainably released from the 0.5BT-, 1BT-, 2BTand 4BT-CTS gels with a concentration range from 1.32 to 8.72 μg/mL for 30 days (Figure S11), which was expected to facilitate skin tissue regeneration considering the favorable effects of Mg stimulation on cell proliferation, differentiation and tissue regeneration as previously reported.44-48 In addition, the embedded B-TiO2-x nanoparticles allowed BT-CTS thermogels to be easily heated up when irradiated by NIR lasers (808 nm). The real-time temperature changes and corresponding infrared thermal images of the BT-CTS thermogels with different B-TiO2-x contents at different power densities of NIR laser irradiation were recorded (Figure 2i-k). All BT-CTS thermogels displayed dramatical temperature increase and apparent color changes, as compared to the negligible temperature elevation for 0T- and 4WT-CTS groups under the same irradiation condition. It was noticed that no obvious visual changes were observed for the 0T-, 0.5WT-, 1WT-, 2WT-, 4WT-, 0.5BT-, 1BT-, 2BT- and 4BT-CTS thermogels after NIR irradiation (808 nm, 0.48W/cm2, 30 min; Figure S7). The photothermal stability of BT-CTS thermogels under photothermal hyperthermia was further confirmed by the negligible changes of temperature profiles for six laser on/off cycles (Figure 2l). Furthermore, the 1O2 generation of BT-CTS thermogels was demonstrated on the DPBF degradation under 10


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NIR irradiation at 808 nm (Figure 2m,n). The DPBF absorption intensity at 410 nm was reduced with the irradiation time for 1BT-CTS thermogels, while no obvious decrement was observed for the 0T- and 1WT-CTS groups. The combined 1O2 generation and heat energy were anticipated to lead to more effective cell destruction considering the synergistic PTT/PDT effect. The injectability of BT-CTS thermogels was investigated by examining the rheological behavior of at 25 ℃ and 37 ℃ (Figure S12). The value of storage modulus (G’, an index of material stiffness) exceeded that of loss modulus (G’’, an index of material viscidity) and a higher value of G’ was observed at 37 ℃ than that at 25 ℃ for both 0T- and 1BT-CTS thermogels, showing a typical behavior of thermosensitive hydrogels.28 Consequently, the BT-CTS thermogels were easily injectable by extrusion through syringes at room temperature and rapidly turned into solid gels at physiological temperature. Notably, the G’ value of BT-CTS thermogels at 37 ℃ increased with the mass content of B-TiO2-x (Figure 2o), confirming that B-TiO2-x nanoparticles could improve the mechanical strength of BTCTS thermogels, which was closely related to the reinforcement function of nanoparticles.49,50 Besides, the value of G’ increased as temperature elevated from 35 ℃ to 55 ℃ (Figure 2p), suggesting the integrity of BT-CTS thermogels could be well maintained even under photothermal hyperthermia. Taken together, the BT-CTS thermogels with easy injectability, excellent thermostability, simultaneous PTT/PDT efficacy endowed them with intriguing potential application in localized tumor therapy. To explore the in vitro tumor therapeutic capacity of BT-CTS thermogels, B16F10 cells (murine melanoma cells) were seeded on the surface of the 0T-, 1WT- and 1BT11


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CTS thermogels or incubated with transwells containing 0T-, 1WT- and 1BT-CTS thermogels. All cells were then irradiated by an NIR laser (808 nm, 0.48 W/cm2, 15 min) and then co-stained with Calcein-AM (green: live cells) and Ethidium homodimer1 (red: dead cells). It was found that B16F10 cells on the surface of 1BT-CTS thermogels or adjacent to the thermogels were almost killed under NIR irradiation (Figure 3a,b). Comparatively, no significant cell damage was observed when the cells were treated by 1BT-CTS thermogels without irradiation and the 0T- and 1WT-CTS thermogels irrespective of irradiation. Quantificationally, the cell viability of B16F10 cells was deceased to 32.0 %, 26.9 %, 18.3 %, and 11.4 % after the treatment of NIRirradiated 0.5BT-, 1BT-, 2BT-, and 4BT-CTS thermogels, respectively (Figure S13). To further test the photodynamic effect on cell viability, B16F10 cells were preincubated with 2,7-dichloro-dihydro-fluorescien diacetate (DCFH-DA) for 30 min and then exposed to NIR irradiation (808 nm, 0.48 W/cm2, 10 min). DCFH-DA was used for detecting ROS generation because it can be oxidized to strong green fluorescent 2,7-dichlorofluorescein (DCF) in the existence of ROS.51 The highly green fluorescence was appeared in the 1BT-CTS+NIR group (Figure 3c) in contrast to negligible fluorescence shown in other control groups, conforming efficient ROS generation and NIR-triggered PDT effect of BT-CTS thermogels. All results above indicated that the BT-CTS thermogels could effectively kill skin tumor cells via simultaneous PTT/PDT effects under the single-wavelength NIR irradiation. Prior to in vivo tumor therapeutic study, the maximum area of tissue defects that could be treated by phototherapy was investigated using three 808-nm lasers with different 12


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laser spot sizes (diameter: 8, 10 and 12 mm) to irradiate the 1BT-CTS thermogels (diameter: 16 mm). The photothermal effective areas (temperature above 45 ℃, which is enough to induce tumor cell death12) were measured when the center maximum temperatures reached 50, 55 or 60 ℃. As shown in Figure S14, larger spot sizes and higher photothermal temperatures induced larger photothermal effective areas with experimental values ranging from 25.22 to 143.41 mm2. The photothermal effective area (> 45 ℃) was calculated as 72.23 mm2 (diameter: 9.6 mm) when the center maximum temperatures reached 55 ℃ for the 1BT-CTS thermogels irradiated by the laser with a spot diameter of 10 mm. Subsequently, a B16F10 tumor-induced wound model was established by creating a full-thickness wound (diameter: 10 mm) at the tumor site (tumor diameter: 4 ~ 5 mm). The tumor-induced wounds were covered by injectable 0T-, 1WT- or 1BT-CTS thermogels (200 μL) and then subjected to the NIR irradiation (808 nm, 0.32 W/cm2, 15 min, spot diameter:10 mm) for four consecutive days (from day 0 to day 3). The wounds covered by 0T-, 1WT- or 1BT-CTS thermogels in absence of NIR irradiation were used as control groups. The temperature in 1BTCTS+NIR group rapidly increased and exceeded 50 ℃ as compared to negligible temperature elevation of 0T-CTS+NIR and 1WT-CTS+NIR groups (Figure 3d,e). Notably, the center temperature of the tumor-induced wounds treated by 1BT-CTS thermogels reached approximately 55 °C, while the temperature of the surrounding healthy tissues was less than 45 °C, indicating that BT-CTS thermogels could kill tumor cells efficiently under the NIR irradiation and avoid damage to surrounding healthy tissues. Tumor growth in 1BT-CTS+NIR group was significantly suppressed after four 13


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days of phototherapy, whereas the tumor volumes of other six groups boosted in an uncontrollable manner (Figure 3f and Figure S15). The photographs of skin tumors from all groups on day 14 (Figure 3g) further conformed the excellent in vivo anticancer effect of BT-CTS thermogels, which should be ascribed to both the NIRinduced hyperthermia and generation of ROS. The tumor-induced wound healing process was tracked for 2 weeks and it was found that the skin wound treated by the NIR-irradiated 1BT-CTS thermogel gradually closed without obvious tumor reoccurrence within 14 days (Figure 4a). However, the wounds hardly healed with the uncontrollable tumor volumes in other groups. The skin tumors and surrounding healthy tissues were resected for cutaneous biopsies (Figure 4b). The primary skin wound at the tumor sites treated by NIR-irradiated 1BT-CTS thermogels was healed as compared to the unclosed skin defects in other six groups. The tumorinduced wound beds were further stained with hematoxylin and eosin (H&E) for histologic analysis (Figure 4c). In sharply contrast to chaotic organization and irregular tumor vascularization beneath the incomplete epidermis observed in other groups, the newly-formed tissue from 1BT-CTS+NIR group highly resembled the normal skin with aligned tissue architectures and regular capillaries, which verified our assumption of firstly eradicating the tumor cells via NIR-triggered phototherapy at early stage (from day 0 to day 3 in present study) and thereafter repairing the cutaneous defects stimulated by BT-CTS thermogels at late stage (from day 4 to day 14 in present study) for healing skin tumor-induced wounds. Successful wound healing is a complicated and well-orchestrated process, 14


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necessitating the collaboration of multiple cell types such as macrophages, keratinocytes, fibroblasts, endothelial cells and so on.52,53 The infiltration, migration, proliferation and differentiation of these cells will result in a timely inflammatory response, succedent new tissue regeneration and eventually wound closure. Keratinocytes involve in restoring the epidermal barrier while fibroblasts and endothelial cells are crucial for the formation of extracellular matrix (ECM) and blood vessels.52, 54 In present study, two typical cell lines, human dermal fibroblasts (HDFs) and human umbilical vein endothelial cells (HUVECs) were employed to investigate the intrinsic tissue regenerative ability of BT-CTS thermogels in vitro. HDFs and HUVECs were seeded on the BT-CTS thermogels and incubated for 24 h. Live/dead assay was performed to detect the cell viability on the thermogel surface, where nearly no dead (red) cells could be observed for all thermogels and the stronger green fluorescence indicated higher cell activity for BT-CTS groups as compared to WT-CTS groups (Figure 5a and Figure S16). In addition, both HDFs and HUVECs proliferated well within 5 days when incubated with the transwells loaded with 0T-, 0.5WT-, 1WT-, 2WT, 4WT-, 0.5BT-, 1BT-, 2BT-, and 4BT-CTS thermogels (Figure 5b,c). Notably, 1BT- and 2BT-CTS thermogels even promoted the proliferation rates of HUVECs (~ 124 % and 144 %, respectively on Day 5) as compared to the blank control (100 %). The proliferation of HDFs was also accelerated when cultured with 0.5BT- and 1BTCTS thermogels for 5 days (~ 130 % and 141 %, respectively on Day 5). The effect of BT-CTS thermogels on cell migration of HDFs and HUVECs was explored using an in vitro scratch assay (Figure S17 and S18). It was found that HDF migration was 15


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stimulated by 1BT- and 2BT-CTS thermogels and the artificially-created wounds completely healed in the 1BT-CTS group after 24 h. Similarly, the cell migration of HUVECs treated with 1BT-CTS thermogels was significantly accelerated within 18 h in comparison with other groups. The generation of new blood vessels or angiogenesis is a critical process during wound healing mainly because blood vessels are required for delivering nutrients and oxygen to the skin cells at wound sites.53-56 The formation of tube-like structures is known as an essential step in angiogenesis.54,57 In this study, a tube formation assay using ECMatrixTM gels was conducted to evaluate the in vitro angiogenic abilities of HUVECs when treated with ionic extracts from the BT-CTS thermogels. As shown in Table S1, there was no significant difference in Ti and Ca ion concentrations among all extracts while higher Mg ion concentrations were observed in the BT-CTS extracts (i.e., 67.43 and 74.06 μg/mL for 1BT- and 2BT-CTS vs. 65.27 μg/mL for pure media). After 4 h of incubation, 1BT-CTS extract exhibited the highest stimulatory effect on the formation of capillary-like polygon networks (Figure S19). Previous studies have shown increased cellular viability and differentiation of bone marrow stromal cells44, 46, 58, vascular smooth muscle cells59 and human tonsil-derived mesenchymal stem cells60 under the stimulation of Mg ions at low concentrations (

Thermogels for Cutaneous Tumor

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