Multi-functional bismuth-doped bioglasses: combining bioactivity and photothermal response for bone tumor treatment and tissue repair

Treatment of large bone defects derived from bone tumor surgery is typically performed in multiple separate operations, such as hyperthermia to extinguish residual malignant cells or implanting bioactive materials to initiate apatite remineralization for tissue repair; it is very challenging to combine these functions into a material. Herein, we report the first photothermal (PT) effect in bismuth (Bi)-doped glasses. On the basis of this discovery, we have developed a new type of Bi-doped bioactive glass that integrates both functions, thus reducing the number of treatment cycles. We demonstrate that Bi-doped bioglasses (BGs) provide high PT efficiency, potentially facilitating photoinduced hyperthermia and bioactivity to allow bone tissue remineralization. The PT effect of Bi-doped BGs can be effectively controlled by managing radiative and non-radiative processes of the active Bi species by quenching photoluminescence (PL) or depolymerizing glass networks. In vitro studies demonstrate that such glasses are biocompatible to tumor and normal cells and that they can promote osteogenic cell proliferation, differentiation, and mineralization. Upon illumination with near-infrared (NIR) light, the bioglass (BG) can efficiently kill bone tumor cells, as demonstrated via in vitro and in vivo experiments. This indicates excellent potential for the integration of multiple functions within the new materials, which will aid in the development and application of novel biomaterials.


S1 Sample preparation
Glass samples of Bismuth (Bi)-doped germanate, silicate and phosphosilicate were prepared by a technique of melting and quenching. As for raw materials, powders of GeO2 (99.99%), SiO2 (99.99%), Bi2O3 (99.99%), P2O5 (99.8%) and analytical grade Na2CO3, CaCO3, Al(OH)3 were employed. Batches of 30 g were prepared for each composition by homogeneously mixing in an agate mortar. These batches were subsequently melted in corundum crucibles in air, and then cast quickly onto a stainless steel plate and pressed immediately with another steel plate to increase cooling rate to prevent possible crystallizations. The glass samples were cut and polished into 10 mm×10 mm×1 mm for consequent measurements. Detailed nominal compositions and melting conditions are summarized in Table S1. Glass samples are coded within column 2 as presented in Table S1. For instance, G5AxB means (95-x) GeO2-5 Al2O3-x Bi2O3 (x = 0.05, 0.5, 1.5, 2, 4, 6, 10 mol%). All the glass samples are bubble-free. The sample without Bi appears colorless. Generally, the sample color becomes deeper as the Bi content increases. For instance, the germanate glass sample is purple-red as x= 0.05, and it becomes reddish-brown and even deep reddish-brown as x = 2 and x = 10, respectively. Similarly to Bi-doped germanate samples, Bi-doped silicate glass shows as reddish-brown while Bi-doped phosphosilicate is light brown. A portion of 5 g of glass samples were manually ground into a powder in an agate mortar. A vertical planetary ball mill (XQM systems, Changsha, China) was then used for further milling, and it employed stainless steel containers and balls of hardened steel with a diameter of 70 mm. The glass powders were milled at a rotation frequency of 350 min -1 for 12 h in dry state. From the resulting powders, the fraction with diameter less than 400 mesh was selected through sieving for consequent biological experiments.

S2 Cell Culture
Two typical representatives of normal cells such as mouse fibroplast cell line (L929) and murine pre-osteoblast cells (MC3T3-E1), and two types of tumor cells for instance rat osteosarcoma-derived cells (UMR106) and human osteosarcoma line cells (U2OS) were purchased from the Type Culture Collection of the Chinese Academy of Sciences. They were dispersed first in a balanced salt solution containing 0.25 % trypsin and 0.04% EDTA, and separated after centrifugation, and eventually incubated at 37 °C in an atmosphere of 5% CO2 and 95% air within Dulbecco's minimum essential medium (DMEM, Gibco, USA) into which 10 % fetal bovine serum (FBS, Gibco, USA), 100 U/mL penicillin and 100 mg/mL streptomycin were added.

S3 In vitro biocompatibility of Bi-doped BGs
Bi-doped phosphosilicate glasses were first sterilized at 120 o C for 20 min, and then put into 24-well plates. Each separated well of the plates was filled by 500 μL DMEM containing L929, MC3T3-E1, U2OS or UMR106 cells in a density of 1×10 4 cells/cm 3 , and they were co-cultured at 37 °C in 5% CO2/95% air for 24 h. 40 μL Phosphate buffer saline with 5 mg/mL methylthiazolyldiphenyl tetrazolium bromide (MTT) was added afterwards into each well, and the sample plates were incubated once again for 4 h at 37 o C. Succinate dehydrogenase produced by these live cells reacted with MTT and led to the formation of blueish violet formazan within cells.
The precipitated formazan was dissolved with 400 μL of dimethyl sulfoxide (DMSO) for consequent measurements on optical density at 490 nm by a microplate reader (Thermo, Multiskan GO). The values were averaged from three independent experiments.

S4 In vitro mineralization of hydroxyapatite on the surfaces of Bi-doped BGs in simulated body fluid (SBF)
In vitro mineralization processes of hydroxyapatite were simulated in SBF solution on the surfaces of Bi-doped BGs. For this, the polished glass samples were further cut into the size of 5 mm× 5 mm×1 mm. Six blank samples of every glass composition were soaked separately in 10 ml of SBF (pH 7.4) for 0, 3, 7, 12, 18 and 24 days at 37 o C and 30% relative humidity in temperature-and humidity-controlled chambers (K-Sun Technology, Guangdong, China). The SBF solution was renewed every other day. After the specified immersion periods, the immersed glass samples were removed from the SBF solution, gently rinsed with distilled water, and dried at room temperature.
Morphology and composition of the generated crystals on the surface were characterized via a field emission scanning electron microscopy (Merlin-SEM, Zeiss, Germany) equipped by an energy dispersive X-ray spectroscopy (EDS, Oxford, England). The crystalline phases were identified by X-ray diffraction (X'Pert Pro, Panalytical, Netherlands).

S5 Adhesion, proliferation, differentiation and mineralization of murine pre-osteoblast cells on Bi-doped BGs
Murine pre-osteoblast (MC3T3-E1) cells were chosen as model osteoblast cells to study the adhesion, proliferation, differentiation and mineralization of the cells on Bi-doped BGs. Similarly to the experiments on in vitro biocompatibility, the cells of MC3T3-E1 were cultured in DMEM for 0, 1 and 3 days, respectively, with glass samples S6PyB where y = 0, 1, 2 mol% (Table S1).
Afterwards, DMEM was removed from the culture wells, and the wells were gently rinsed three times with phosphate buffer saline (PBS) to remove residual DMEM. The wells were refilled in consequence with 500 μL of 2.5% glutaraldehyde solution to fix the cells on the sample surfaces.
The samples were washed 3 times with PBS, and dehydrated in a series of solutions with the increase of ethanol content from 30 %, to 50, 70, 80, 90, 95 and 100 %. They were eventually coated with gold for SEM images (EVO18, Zeiss, Germany) to monitor the morphological change and the cell adhesion on sample surfaces.
Glass samples S6PyB (y=0, 1, 2 mol %) were put in the 24-well plates which were filled by 500 μL DMEM with a density of 1×10 4 MC3T3-E1 cells per cm 3 . Osteogenic differentiation of MC3T3-E1 cells was induced by addition of 1 % osteogenesis revulsant into DMEM. The osteogenesis revulsant was prepared with 100 mmol/L dexamethasone, 0.05 mmol/L ascorbic acid and 10 mmol/L Na-β-glycerophosphate. As a reference, a control group was prepared in parallel without glass samples. 500 μL triton X-100 was added into each well after removal of DMEM and reacted overnight with MC3T3-E1 cells to release alkaline phosphatase (ALP). 50 μL p-Nitrophenyl phosphate was subsequently added to the solution and reacted with ALP and therefore produced p-nitrophenol. For Day 7 and Day 14 samples, ALP activities were determined with a colorimetric assay 1, 2 by monitoring the optical density at 405 nm. Each experiment was repeated three times and ALP activity was averaged accordingly.
In vitro mineralization of osteoblast cells was evaluated by a technique of Alizarin Red S staining 3 . Figure S1. Scheme for quantification of the PT effect.