A tissue-engineered therapeutic device inhibits tumor growth in vitro and in vivo

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A tissue-engineered therapeutic device inhibits tumor growth in vitro and in vivo Ming Sun a,⇑, Miao Wang a, Muwan Chen b,⇑, Frederik Dagnaes-Hansen c, Dang Quang Svend Le b, Anette Baatrup a, Michael R. Horsman d, Jørgen Kjems b, Cody Eric Bünger a a

Orthopaedic Research Lab, Aarhus University Hospital, Aarhus C, Denmark Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus C, Denmark Department of Biomedicine, Aarhus University, Aarhus C, Denmark d Experimental Clinical Oncology Department, Aarhus University Hospital, Aarhus C, Denmark b c

a r t i c l e

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Article history: Received 10 November 2014 Received in revised form 20 January 2015 Accepted 3 February 2015 Available online xxxx Keywords: Tissue engineered therapeutic device Local drug delivery Bone metastases Tumor inhibition Bone substitute

a b s t r a c t Bone metastasis is one of the leading causes of death in breast cancer patients. The current treatment is performed as a palliative therapy and the adverse side effects can compromise the patients’ quality of life. In order to both effectively treat bone metastasis and avoid the limitation of current strategies, we have invented a drug eluting scaffold with clay matrix release doxorubicin (DESCLAYMR_DOX) to mechanically support the structure after resecting the metastatic tissue while also releasing the anticancer drug doxorubicin which supplements growth inhibition and elimination of the remaining tumor cells. We have previously demonstrated that this device has the capacity to regenerate the bone and provide sustained release of the anticancer drug in vitro. In this study, we focus on the ability of the device to inhibit cancer cell growth in vitro as well as in vivo. Drug-release kinetics was investigated and the cell viability test showed that the tumor inhibitory effect is sustained for up to 4 weeks in vitro. Subcutaneous implantation of DESCLAYMR_DOX in athymic mice resulted in significant growth inhibition of human tumor xenografts of breast origin and decelerated multi-organ metastasis formation. Fluorescence images, visualizing doxorubicin, showed a sustained drug release from the DESCLAYMR device in vivo. Furthermore, local use of DESCLAYMR_DOX implantation reduced the incidence of doxorubicin’s cardio-toxicity. These results suggest that DESCLAYMR_DOX can be used in reconstructive surgery to support the structure after bone tumor resection and facilitate a sustained release of anticancer drugs in order to prevent tumor recurrence. Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

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1. Introduction As many as 70% of breast cancer patients exhibit bone metastases at autopsy [1]; the development of distant metastases, including bone cancer, is the leading cause of death in breast cancer patients. Currently, the treatment of bone metastases comprises local therapies, including radiation therapy and surgery, which are performed mainly as palliative therapies [2]. The standard care for patients after tumor resection surgery is radiation and/or systemic chemotherapy [3]. The drawback of systemic administration

⇑ Corresponding authors at: Orthopaedic Research Laboratory, Aarhus University Hospital, Noerrebrogade 44, Building 1A, DK-8000 Aarhus C, Denmark. Mobile: +45 5268 6688 (M. Sun). Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wiedsvej 14, DK-8000 Aarhus C, Denmark. Mobile: +45 4125 4073 (M. Chen). E-mail addresses: [email protected] (M. Sun), [email protected]. dk (M. Chen).

of high dose anticancer drugs is their toxicity to healthy cells, resulting in adverse side effects including severe immune suppression, nephrotoxicity, and cardiotoxicity [4,5], all of which can severely compromise the patients’ quality-of-life [3,6]. Furthermore, systemically administering drugs limits their ability to reach and penetrate neoplastic cells distant from tumor vessels [7]. In addition, their short half-life reduces their bioavailability and subsequent effectiveness in eliminating tumor cells [8]. To avoid all the limitations of systemic chemotherapy, local use of chemotherapeutic drugs has been introduced. Chemotherapeutic drugs incorporated into scaffolds have been widely investigated [9,10]. At present, local sustained drug release from an implanted device at the resected tumor site is a more realistic approach to be implemented. The GliadelÒ Wafer, which is used in the treatment of brain tumors, is an early example of a drug delivery system in clinical use [11]. Although the wafer had no significant impact on the survival of treated patients [12], patients reported a higher quality-of-life compared with those treated by conventional

http://dx.doi.org/10.1016/j.actbio.2015.02.004 1742-7061/Ó 2015 Published by Elsevier Ltd. on behalf of Acta Materialia Inc.

Please cite this article in press as: Sun M et al. A tissue-engineered therapeutic device inhibits tumor growth in vitro and in vivo. Acta Biomater (2015), http://dx.doi.org/10.1016/j.actbio.2015.02.004

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systemic chemotherapy [13–15]. In the case of breast cancer bone metastases, tumor resection must be followed by bone grafts or artificial substitutes in place of the resected tissue in order to provide immediate mechanical support. In recent years, the increasing interest in fabrication of drug-eluting bone tissue engineering scaffolds is a logical evolution because such scaffolds provide an approach that conventional medical practice does not offer [16–18]. We recently reported a drug eluting therapeutic device DESCLAYMR, which could fulfill the requirements for both bone tissue engineering and local sustained release of an anticancer drug in vitro [19]. DESCLAYMR scaffold comprises a load bearing macroporous polycaprolactone (PCL) component made by fused deposition modeling embedded with a microporous foam with chitosan and montmorillonite clay made by thermally induced phase separation. With the ability to provide biomechanical support, the PCL scaffold has been reported to possess highly promising potential as a substitute for bone graft [20–22]. A cumulative strategy for treating breast cancer bone metastasis is to devise a multifunctional implant to support the skeletal structure and combine it with local, controlled release of an effective drug to prevent cancer recurrence [9,17]. In this study, we have investigated the release profile and tumor inhibitory effect of scaffolds loaded with doxorubicin in human breast cancer cell lines, examined the tumor growth and multi-organ metastases inhibition of DESCLAYMR_DOX implantation in tumor-bearing mice, and tested in vivo doxorubicin release profiles.

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2. Materials and methods

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2.1. DESCLAYMR scaffold

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Components: The nanoclay (sodium montmorillonite) was Cloisite Na+, Lot: 07F28GDX-008 (Southern Clay Products, Inc., Moosburg, Germany). The chitosan was Chitopharm M with 75–85% degree of deacetylation (Cognis, Florham Park, NJ). Polycaprolactone (MW = 50 kDa) was from Perstorp (Cheshire, UK). The b-TCP nanocrystals were Lot: TCPCH01 (Berkeley Advanced Biomaterials, Inc., Berkeley, CA). The DESCLAYMR scaffold was produced as previously described [19]. Briefly, 3D PCL printed scaffolds (d = 4 mm, h = 2 mm) were immersed coated with 1% w/v chitosan in 1 vol.% acetic acid solution, containing chitosan modified montmorillonite clay (weight ratio to chitosan is 1:10, clay final concentration to chitosan solution is 0.9 mg/ml), and tricalcium phosphate (weight ratio to chitosan is 1:20). Scaffolds were freeze-dried and then neutralized in 70% Ethanol containing 0.4 M NaOH and then rinsed in PBS three times and freeze-dried again. Scaffolds before drug loading were stored at room temperature in a desiccator.

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2.2. Drug

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Doxorubicin hydrochloride (Sigma–Aldrich, Denmark) was suspended in Phosphate-buffered saline (PBS) at a concentration of 4 mg/ml (DOX). Fifteen microliters of DOX at two concentrations (4 mg/ml and 2 mg/ml) was added on the surface of the DESCLAYMR scaffold (high amount group: 60 lg/scaffold; low amount group: 30 lg/scaffold) and air-dried overnight (DESCLAYMR_DOX). Scaffolds with drug loading were kept at 20 °C without light exposure.

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2.3. Cell line

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The MDA-MB-231-luc-D3H2LN BiowareÒ (231-luc) (PerkinElmer Inc., U.S.) is a luciferase- expressing cell line. This cell line

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has enhanced tumor uptake and metastatic potential compared to the parental cell line. Cells were maintained in Eagle’s Minimum Essential Medium (EMEM) (ATCCÒ 30-2003™) with 10% fetal bovine serum (FBS) (ATCCÒ 30-2020™) and incubated at 37 °C in 5% CO2 in a humidified incubator.

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2.4. Doxorubicin release profile

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The release profile of DOX from the scaffold was determined by incubating four scaffolds in 1.2 ml of sterile PBS (pH = 7.4) at 37 °C (n = 4) in a sterile incubator for different time intervals. Before the release study, scaffolds were washed in PBS to remove the free DOX. At each time point, 1.2 ml of solution was collected and replaced with 1.2 ml of fresh PBS to avoid the degradation of DOX. The fluorescence intensity of DOX in the buffer solution was quantified with a Victor 1420 multilabel counter (Wallac, Waltham, MA) with excitation at 405 nm and emission at 615 nm. The concentrations of DOX released in the solutions were calculated according to the calibration curve of DOX in PBS and the cumulative release rates were calculated afterward.

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2.5. Cytotoxicity of drug-release solution

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Viability of the cells cultured in released solution from different time points for 3 days was measured using the XTT assay from Roche (Cat. No. 11465015001, Roche) in accordance with the manufacturer’s instructions. Briefly, the culture medium was removed and 150 ll XTT solution was added. After incubating for 18 h, 100 ll XTT solution was added to a 96-well plate. The orange formazan dye, formed from the conversion of the XTT yellow tetrazolium salt by metabolically active cells, was measured at 450 nm using a Victor 1420 multilabel counter. Cell numbers were estimated with methylene blue staining read on a Victor 1420 multilabel counter.

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2.6. Human xenograft tumors

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All animal studies, including maintenance and determination of experimental endpoints, were performed according to the Danish Experimental Animal Inspectorate. The experimental procedures were shown in Fig. 1. Ten to fourteen week old female BALB/ cATac-nude (Taconic Farms Inc., Germantown, USA) mice were used for implantation of tumor cells. The animals received one subcutaneous injection of 3  106 231-luc cells in each side of the hind flanks. Tumor volumes were measured in two dimensions twice a week after implantation and the tumor volumes (Vt) [(L*W2)/2] were calculated from caliper measurements. When the tumors reached an average volume of 200 mm3, treatment with subcutaneous implantation of DESCLAYMR_DOX (60 lg/scaffold), which was selected for the in vivo study according to the results from in vitro cytotoxicity study, and subcutaneous injection of the same dose of DOX was initiated. The sites of DESCLAYMR implantation and DOX injection were located subcutaneously, at a distance of 5 mm to the tumors. The experimental endpoint was reached when ulcerations of the tumors occurred, or weight losses were >10% of mouse bodyweight measured before any procedures, or averaged tumor volumes in the group were >1500 mm3, or skin ulcerations occurred due to the subcutaneous use of DOX in some cases (INJECTION_DOX group).

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2.7. Imaging and quantification of bioluminescence data

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The mice were anesthetized with a mixture of 3.5% isoflurane/ air using an Inhalation Anesthesia System. D-Luciferin (Caliper Life Sciences, Hopkinton, MA) was intraperitoneally injected at 150 mg/kg mouse body weight. Ten minutes after D-luciferin

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Fig. 1. In vivo experimental schema: day 1: the mice were subcutaneously implanted with human breast cancer cell line: MDA-MB-231-D3H2LN. Day 13: the averaged tumor volumes reached 200 mm3 and treatment with subcutaneous implantation of DESCLAYMR_DOX and subcutaneous injection of the same dose of DOX (INJECTION_DOX) was initiated. Mice without any treatments were set up as controls (BLANK_CONTROL). Day 27: all the mice were euthanized. Tumors and organs (hearts, lungs, stomachs, spleens/pancreas, livers and kidneys) were collected, imaged and fixed for histologic examination.

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injection, the mice were imaged with a Xenogen IVIS Spectrum imaging platform (PerkinElmer, MA) with continuous 2.5% isoflurane exposure. Imaging variables were maintained for comparative analysis. A region of interest (ROI) was manually selected over relevant regions of signal intensity. The intensity was recorded as surface radiance (photons/s/cm2/sr) within a ROI. At the end of the experiments, the animals were euthanized by cervical dislocation ten minutes after D-Luciferin was intraperitoneally injected in order to allow for assessment of multi-organ metastases using ex vivo bioluminescence. The organs of interest were removed, arranged on black, bioluminescence-free paper, and ex vivo imaged with the IVIS scanner within 30 min.

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2.8. Imaging and quantification of fluorescence data

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The mice were imaged with the IVIS scanner at different time points in order to detect of doxorubicin fluorescence, either loaded into DESCLAYMR scaffolds or injected subcutaneously. DOX fluorescence was captured on a CCD camera (excitation 430–465 nm/ emission 540–640 nm) and analyzed after spectral unmixing. A ROI was manually selected according to the size of the scaffold or injection area; the area of the ROI was kept constant in the course of the experiments. The intensity was recorded as radiant efficiency ([photons/s]/[lW/cm2]) within a ROI.

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2.9. Histologic and immunohistochemical examination

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The dissected tumors and organs were fixed in 70% ethanol prior to processing. Tissues were processed in a series of increasing ethanol concentrations and embedded in paraffin wax. Fivemicrometer sections were cut and stained with hematoxylin–eosin (H&E). Immunostaining for firefly luciferase was also done. Briefly, sections were deparaffinized in xylene, rehydrated in decreasing ethanol to H2O, microwaved for ten minutes in 10 mmol/l citrate buffer (pH 6.0) at 95–99 °C for antigen retrieval, and finally endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Endogenous biotin was blocked using a streptavidin kit (Molecular Probes, cat No. E-21390). Nonspecific binding was abolished with 10% bovine serum albumin (BSA) for 20 min. Tissue sections were incubated overnight at 4 °C with a 1:500 dilution of 10 mg/ml biotin-conjugated goat anti-firefly luciferase antibody (Abcam, Hartford, CT). After three PBS washes with 0.1% Tween for 15 min, sections were incubated with 1:300 Stept-Avidin HRP

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conjugated (Dako, cat No. P0397). After three PBS washes, sections were incubated in 3-Amino-9-Ethylcarbazole (Sigma, cat No. A6926) for 25 min. Finally, sections were washed in distilled water to terminate the reaction, and the nuclei were stained with Mayer’s hematoxylin for 30 s, washed in distilled water and mounted.

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Hearts were fixed in 70% ethanol, embedded in paraffin, cut in 5 lm slices, and stained with H&E. Fibrillar collagen in tissue sections was detected by 0.1% picrosirius red staining (Sigma–Aldrich Inc., Denmark). After staining, the sections were kept in the dark and incubated for 30 min. They were then rinsed with distilled water, dehydrated, and mounted with permount. The sections were visualized and photographed by a camera on an Olympus microscope with 200 magnification.

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2.11. Statistical analysis

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Unless otherwise stated, each sample was assayed in quadruplicate. Each in vitro experiment was repeated three times and each in vivo experiment was repeated twice. Unless otherwise noted, data are presented as mean ± SEM; a Student’s t test (unpaired and two-tailed) was used to compare two groups of independent samples. Correlation of DNA quantification of tumor cells and concentration of DOX was analyzed using a Spearman Rank Correlation Test. Results were subjected to statistical analysis using STATA v11.0 software (Stata Corporation, Lakeway Drive, TX).

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3. Results

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3.1. In vitro release profile and cytotoxicity of DOX from DESCLAYMR_DOX

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At the first 24 h, DOX was released 14.1 ± 2.0% of total amount of drug in the high amount group (contained 60 lg DOX/scaffold at the beginning of the release assay), while 27.4 ± 7.2% DOX was released from the low amount group (contained 30 lg DOX/scaffold at the beginning of the assay). From day 1 to 4 weeks, around 15% and 27% DOX were released from the high and low amount groups. By day 84, 32.3 ± 5.5% DOX was released from the high amount group and 58.7 ± 17.6% DOX was released from the low

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amount group cumulatively (Fig. 2A). In the aspect of released DOX concentrations, 15.2 ± 1.8 lg/ml and 9.8 ± 0.7 lg/ml DOX was released from the high and low amount groups within the first day. At 84 days, 3.5 ± 1.2 lg/ml and 1.6 ± 0.5 lg/ml DOX was release in high and low amount groups, respectively (Fig. 2B). We used these DOX release solutions to test the cytotoxicity effect on breast cancer cells. Results showed that the release solution from no DOX loaded scaffold had no cytotoxic effect on the 231-luc cells at all the time points, and the cells responded in a dose-dependent manner to the DOX (p < 0.001; Spearman Rank Correlation Test). As the concentration of DOX in released solution at serial time points was decreased, the cell viability (Fig. 3A) and the number of viable cells (Fig. 3B) increased in both groups. From the high amount group, a significant decrease (compared with the control) in viable cell number treated with released solution at 1 day, 3 days, 1 week, 2 weeks, 3 weeks, and 4 weeks was observed (p < 0.05). Significant cell growth inhibition was observed for 2 weeks in the low amount group (p < 0.05). Thus, we continued with the high amount group for the in vivo study.

3.2. In vivo inhibition of tumor growth and release profile of DOX from DESCLAYMR scaffold Significant delay in tumor growth was observed after 8 days of treatments, either with subcutaneous implantation of DESCLAYMR_DOX or subcutaneous INJECTION_DOX compared with BLANK_CONTROL (p = 0.0033; 0.0385, respectively; Fig. 4A).

Measuring tumor burden with an IVIS scanner revealed that DESCLAYMR_DOX showed greater tumoricidal effects compared to INJECTION_DOX (Fig. 4B and C). We measured the relative concentrations of DOX in the treatment area by detecting DOX fluorescence (Fig. 5). For the DESCLAYMR_DOX group, there was an increased DOX concentration during the first 4 days, followed by gradually decreased DOX until day 14. For the INJECTION_DOX group, there was a burst release four hours after treatment followed by a fast decrease on day 1. There was no detectable DOX after 4 days post treatment in the INJECTION_DOX group (Fig. 5A and B). DOX concentration in the treatment area in the DESCLAYMR_DOX group was significantly different at all examined time points compared with the INJECTION_DOX group (p < 0.05).

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3.3. Systemic antitumor effect and other adverse side effects

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Ex vivo bioluminescence revealed multi-organ metastases, which were confirmed later by H&E- and immunohistochemical staining for tumor tissue and organs followed by application of anti-luciferase antibody. Fig. 6A showed examples of metastasis identified in different organs from the three groups (BLANK_CONTROL, INJECTION_DOX and DESCLAYMR_DOX). Tissue sections were scored as positive or negative based on the presence or absence of detectable metastases. Mouse with positive tissue sections was diagnosed as metastases in related organs. Multi-organ metastasis incidence in each group was calculated by number of

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Fig. 2. Release profile of doxorubicin from DESCLAYMR scaffolds loaded with different DOX concentrations. (A) Cumulative DOX release (%) from the high amount group and the low amount group at 37 °C in PBS (pH 7.4) for 12 weeks. (B) DOX release concentration at each time point in both groups. Data are presented as mean ± SEM. n = 6 in each group.

Fig. 3. Tumor inhibition of released solution at serial time points in human breast cancer cells: (A) cell viability by XTT assay, normalized to control (media alone). Cells were cultured in released solution from serial time points for 3 days. All values are reported as mean ± SEM (n = 4). ⁄p < 0.05. (B) Viable cell number by methylene blue staining assay, normalized to control. Cells were cultured in released solution from serial time points and media for 3 days. All values are reported as mean ± SEM (n = 4). ⁄p < 0.05.

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Fig. 4. Average tumor volumes in treatment groups increased more slowly than in the control group from 8 days post treatment: (A) BLANK_CONTROL group reached tumor volume endpoint (1500 mm3) at 27 days, with comparative tumor volume significantly reduced in the DESCLAYMR_DOX (p = 0.0007 by Student’s t test) and BLANK_CONTROL groups (p = 0.0264 by Student’s t test) at the same time. DESCLAYMR_DOX, n = 10; INJECTION_DOX, n = 6; BLANK_CONTROL, n = 10. (B) Biweekly quantification of bioluminescence showed decelerated tumor growth in the DESCLAYMR_DOX and INJECTION_DOX groups compared with the BLANK_CONTROL group. Mice receiving DESCLAYMR_DOX showed greater tumor growth inhibition compared to mice receiving INJECTION_DOX. Data are presented as mean ± SEM. n = 6 in all groups. (C) Representative bioluminescent images visualizing viable tumor cells showed a tumor growth inhibition in the presence of DESCLAYMR_DOX compared with other groups.

Fig. 5. In vivo drug release profiles of DOX from DESCLAYMR scaffolds: (A) fluorescence quantification showed an increased fluorescent intensity of DOX in the DESCLAYMR_DOX group in the first 4 days, reaching a maximum at day 4 post-treatment, while a fast decrease of fluorescent intensity of DOX was detected in the INJECTION_DOX group with no detectable DOX after 9 days post-treatment. Data are presented as mean ± SEM. n = 6 in all groups. (B) Representative fluorescence images visualizing DOX either loaded into the DESCLAYMR scaffold (DESCLAYMR_DOX) or injected subcutaneously (INJECTION_DOX) 2 weeks after treatment. DOX loaded into the DESCLAYMR scaffold was detectable on day 14 after implantation compared with undetectable DOX in INJECTION_DOX group.

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animal with metastasis-diagnosis divided by total animal number in this group. Data are shown in Fig. 6B and there was an increased development of multi-organ metastases in the BLANK_CONTROL group compared with mice receiving DESCLAYMR_DOX or INJECTION_DOX (Fig. 6B). The multi-organ metastases incidence was particularly higher in the liver and stomach in the INJECTION_DOX group compared with the DESCLAYMR_DOX group. Finally, sections of mouse heart were stained with H&E to visualize the change in morphology. Better myocardial structures were found in mice receiving DESCLAYMR_DOX compared with INJECTION_DOX (upper panel, Fig. 7). Picrosirius red staining was used to evaluate fibrosis in heart sections. Positive picrosirius red stains were absent in the DESCLAYMR_DOX group and the BLANK_CONTROL group while presented in the INJECTION_DOX group (lower panel, Fig. 7).

4. Discussion

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Here we report that a drug-eluting scaffold with clay matrix release (DESCLAYMR) device, when loaded with the anti-cancer drug doxorubicin (DESCLAYMR_DOX), has an inhibitory effect on human breast cancer cell lines in vitro and on tumors in a xenograft model. In comparison to mice which received subcutaneous injection of the same dosage of DOX (INJECTION_DOX), subcutaneous DESCLAYMR_DOX-treated mice showed more sustained tumor growth inhibition and no adverse side effects such as skin ulceration and cardiotoxicity. With regard to the drug release kinetics, both the high amount group and low amount group showed an observable burst release of DOX in the first 24 h, followed by a four-week sustained release and a small amount of DOX release until 12 weeks. The initially

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Fig. 6. Decreased multi-organ metastases in mice treated with DESCLAYMR_DOX: (A) representative examples of metastases in liver, kidney, spleen/pancreas and stomach are shown using ex vivo bioluminescence, with confirmation by H&E- and anti-luciferase immunostaining (representative images shown). (B) Multi-organ metastasis incidences in different treatment groups: metastases were identified following the aforementioned procedures in organs of the Mda-Mb-231-LUC tumor model in Balb/c nude mice receiving DESCLAYMR_DOX (n = 10), INJECTION_DOX (n = 6) or no treatment (BLANK_CONTROL, n = 10).

Fig. 7. Absence of fibrosis in the hearts of mice receiving DESCLAYMR_DOX implantation: H&E staining showed better myocardial structure preservation in mice treated with DESCLAYMR_DOX compared with INJECTION_DOX. Fibroses, stained by picrosirius red, were found in the INJECTION_DOX group while absent in the DESCLAYMR_DOX group and the BLANK_CONTROL group.

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burst release likely originates from the remaining free doxorubicin that was not encapsulated into the nanoclay. The first 4 weeks’ sustained release was from the cationic exchange of Na+/Ca2+ in the PBS with the clay-intercalated DOX. After 4 weeks, the slow release suggested that the clay-DOX interaction might reach the equilibrium of ion exchange. However, this in vitro release profile differed from the previous study by Chen et al. [19]. The reason may be related to the drug loading method employed: rather than encap-

sulating DOX into the clay layers and designing as a clay/DOX carrier, we loaded the drug directly onto the DESCLAYMR scaffold. This drug coating method resulted in the drug loading efficiency is about 50–60% and there is an unavoidable fast release in the first 24 h. Different loading amounts did not necessarily change the release pattern. In order to improve the release profile, we could increase the amount of the clay-intercalated DOX by mixing the DOX into the DESCLAYMR scaffold-manufactured procedure to

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achieve a prolonged sustained release, as our previous paper described. The drug-release kinetics can also be affected by the pH of the medium [23,24]. In the current study, we only tested the drug release in PBS (pH7.4) buffer because we considered this device would be used as an implant at the bone environment, where the pH of the tissues ranges from 7.2 to 7.6. Clay–drug interaction has been widely studied and used to provide a sustained release of drugs in pharmaceutical products [23–31]. From the tissue-engineering point of view, studies show that incorporation of clay with chitosan and hydroxyapaptite improves both mechanical and osteogenic scaffold properties [32,33]. However, most the studies were focusing at using clay or clay composites as a drug carrier for oral drug delivery instead of as a component in an implantable device for local drug delivery [23,24]. Furthermore, attention should be given to the safety of clay minerals since there are reports on the cytotoxic effect of unmodified and organically modified nanoclays in the human hepatic cell line HepG2 [34]. Verma et al. [35] compared the different nanoclays’ toxicity to human epithelial cells and showed that platelet-type nanoclay at 25 lg/ml and tubular structured nanoclay at 250 lg/ml have cytotoxicity. In our device, we assume it is relatively safe since the concentration of nanoclay we used for fabricating our scaffold was 0.9 lg/ml [19]. There are other studies using 3D scaffolds as drug carriers: Choi et al. [36] reported that by encapsulating DOX into the scaffold via nanoparticles, a 3D poly (propylene fumarate) (PPF) scaffold showed a steady drug release rate for the first 24 h. Wang et al. [37] combined DOX-entrapped bisphosphonate-derivatized (BP) liposomes and composite scaffolds made up of collagen and hydroxyapatite (Col/HA) and found that the drugs entrapped in BP-liposomes showed a slower release from the Col/HA scaffolds in 48 h, while unencapsulated drugs and drugs encapsulated in PEG-liposomes displayed rapid release from the scaffolds. However, both of these systems will require extended observation periods in order to conclude anything about sustained DOX release. Results from the XTT assay and Methylene blue assay, which were employed to analyze cytotoxicity of the DESCLAYMR_DOX in vitro, are consistent. They both exhibit the same trend of reacting to the released DOX in a dose dependent manner. The high amount group showed significant cell growth inhibition for up to 4 weeks compared with only up to 2 weeks in the low amount group. A study by Zhu et al. [38] reported that DOX loaded hydrogel could inhibit the growth of human bladder carcinoma EJ cells by 40–50% after 72 h incubation. Defail et al. reported a reduction in viable 4T1 tumor cells was observed by treating the cells with gelatin scaffolds loaded with DOX-encapsulated PLGA for 48 h [9]. Compared with the above studies, the prolonged observation time in our study regarding cytotoxicity predicts a long-term use of this device in tumor inhibition. We further investigated the in vivo release profile as well as the tumor inhibition effect of DOX from the DESCLAYMR scaffold. The local release of DOX can be estimated based on the drug concentration in the area of the injection site and in the scaffold. As the tumors were situated subcutaneously, an in vivo semi-quantitative measurement on relative concentration of DOX was possible. With the same amount of DOX, either loaded in the implanted DESCLAYMR scaffold or subcutaneously injected in a position similar to the implantation, significant variations in detectable fluorescence of DOX between the two administrations were observed. A fast decrease in the INJECTION_DOX group indicates spontaneous diffusion and fast clearance of the drug (Fig. 5). This is in accordance with the tumor inhibition effect illustrated in Fig. 4B. In the DESCLAYMR_DOX group, we found an increase of DOX in the treatment area within the first 4 days, which means that the release of DOX from the device prolongs the presence of DOX at the treatment site to a much larger extent than from the

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INJECTION_DOX group. The sustained release of DOX appeared to result in a stronger inhibition of tumor growth on day 4 and day 9 post treatment compared with the INJECTION_DOX group, although these differences were not statistically significant (Fig. 4B). From the in vitro DOX release profile, we know that the DESCLAYMR scaffold can release DOX in a sustained manner for 4 weeks. The sustained release of DOX was observed as well in vivo, however for ethical reasons we can only observe the animals for 2 weeks. The method we used for measuring in vivo release imposes limitations inasmuch as the IVIS scanner only enables us to measure the surface of the treated area, which gives the relative concentration of local released DOX but not the absolute drug amount. However, as the same restriction applies to the INJECTION_DOX group, we were still able to compare the difference between the two groups. To evaluate tumor growth inhibition from the different treatments, we employed two methods: tumor volumes were measured in three dimensions using a caliper twice a week and tumor burdens were estimated by photon emission rates from the tumors in an IVIS scanner. Current standard for volumetric measurement of xenograft tumors is by external caliper due to several advantages, such as rapidness, non-invasion and inexpensiveness [39,40]. However, it is not precise enough when tumors, especially human xenograft lines transplanted and grown in mice, possess shape changes which can no longer be calculated as an ellipsoid volume [41]. Additionally, intra-observer variation can highly influence the measurements and result in imprecise tumor volume estimation. In this study, tumor volumes were determined independently by two observers to minimize the intra-observer bias and a bioluminescent imaging (BLI) technology was employed to compensate the limitations of caliper-measurement. The BLI method is a direct method to investigate cell proliferation in vivo. Since the reaction that produces photons requires oxygen, ATP, luciferase, and the substrate D-luciferin, BLI only detects vascularized [42], viable tumor cells without actually detecting necrotic or dying cells inside the tumor. Additionally, BLI can be affected by the degree of tumor perfusion and oxygen, therefore possesses inaccurate measurement of a fast-growing tumor, which commonly has areas of hypoxia. In case of our study, it is crucial that we employed two methods to estimate the tumor size with a fast-growing rate and evaluate the reaction of tumor to different treatments. The tumor volume measurement reflects the total volume within the tumor, thus includes both viable cells and areas of necrosis (the necrosis includes both that which occurs as a result of tumor growth and as a result of cell killing by the drug). The BLI method only reflects viable cells. In the tumor volume study, both INJECTION_DOX and DESCLAYMR_DOX significantly inhibited tumor growth when compared to controls. The tumor growth curve for mice receiving DESCLAYMER_DOX was also below that for INJECTION_DOX, but was not statistically significant. With the bioluminescence assay, there was less activity of tumor cells in the INJECTION_DOX group compared to controls and even lower activity in the DESCLAYMER_DOX group, but these trends were not actually significant. Thus, our results are consistent with an antitumor effect of systemically injected DOX and a somewhat larger effect when DOX was administered in the scaffold. Several studies have reported local control of tumors using local treatment device. A study from Manabe et al. [43] reported on a polyurethane-based pouch, which was sutured subcutaneously in tumor-bearing mice and loaded with gemcitabine 3 days after tumor inoculation. Four of six mice treated with loaded devices had no observable tumor mass after 30 days, while the remaining two mice developed tumors at a rate comparable to the control mice. Wolinsky et al. [44] used poly (glycerol monostearate-co-e-caprolactone) films as a controlled, prolonged, and low dose delivery matrix for the potent anticancer agent 10-hydroxycamptothecin (HCPT). These drug-loaded films

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were investigated in a series of studies and found to be highly effective at completely preventing local recurrent tumor growth in several animal models of local tumor recurrence [45,46]. Biodegradable cross-linked chitosan hydrogels loaded with radioactive hydrogels have been introduced as treatment in a mouse mammary tumor model and have been shown to prevent tumor recurrence and metastatic spread [47]. While all of these studies use a local treatment device to treat either micro-size or resected tumors. In our study, the therapeutic device is effective to tumors of average size 200 mm3, indicating that it could be more effective in the treatment of residual disease, especially in tumor-debulking surgeries. Only a few tissue-engineering scaffolds have been investigated for their tumor inhibitory effects. Kutlu et al. [48] reported a chitosan scaffold loaded with antiangiogenic agent, bevacizumab and PLGA nanoparticle carrying drug 5-FU for brain tumor therapy, which inhibited the viability of tumor cells in vitro. Shin et al. [49] composited a Polyphenol bearing cinnamaldehyde scaffold and showed the scaffold had cell growth inhibitory effects on cisplatin-resistant ovarian cancer cells in vitro. A bone void filler material, the biodegradable poly-cyclodextrin functionalized porous bioceramics has been developed to achieve a high local drug concentration which may have better control of residual malignant cells and promote reconstruction of bone defects. It showed a prolonged (up to 24 h) cytotoxic effect while loaded with chemotherapeutic drugs to osteosarcoma cells in vitro [50]. However, none of these drug-delivery devices has been tested in vivo. With an aim toward reducing recurrence rates of spinal metastases after surgeries, further investigation regarding DESCLAYMR_DOX in a breast cancer spinal metastases model needs to be conducted. Metastatic disseminations from primary tumors can reach every organ in the body, which is a great therapeutic challenge in clinics. Current therapies have improved the control of the systemic disease yet consequently enhanced the risks of exposing healthy organs to high doses of toxic chemo-drugs. Local chemotherapy with a lower systemic drug concentration may be one of the solutions. Therefore, it is important to investigate the occurrence of multi-organ metastasis using the treatment of DESCLAYMR_DOX. Inhibition of distal metastases in mice receiving DESCLAYMR_DOX may be a result of lower tumor burdens at the primary tumor site. It is also possible that DESCLAYMR_DOX may play a role as a reservoir for DOX in vivo and the prolonged release in the treatment site may result in a prolonged exposure of DOX to other organs, which will require more investigations such as plasma DOX concentration and/or another animal model of treating metastatic tumor. In the spleen/pancreas, we found more metastases in the DESCLAYMR_DOX group than in the INJECTION_DOX group and no difference in kidney metastases, which will require further investigations that include more animals in the studied groups. The adverse side effects of DOX, especially cardiotoxicity, have been the main obstacle for the use of this drug in clinics. Lower doses of DOX, continuous infusion, or different formulations may be less cardiotoxic [51], which may be assessed by using DESCLAYMR_DOX. The presence of positive fibrosis stains in the INJECTION_DOX group suggests that cardio-toxicity-related doses of DOX may occur during one bolus administration of DOX, while we found no positive fibrosis stain in the DESCLAYMR_DOX and BLANK_CONTROL groups. These observations suggested the DESCLAYMR_DOX provided a relatively low systemic DOX level with no detected cardio-toxicity. A subcutaneous tumor model was used in this study to evaluate the anti-tumor efficacy of the DESCLAYMR_DOX. In this proof-ofprinciple study, we successfully demonstrated the tumor inhibition of using the DESCLAYMR_DOX implantation subcutaneously. In order to achieve the aim of local bone tumor treatment, we need to develop a rodent model with bone metastases for future studies.

Furthermore, scaffolds incorporating with DOX will have effects on the bone regeneration and this requires more investigations. We are currently performing a bone biocompatibility study in a porcine model with humerus defect to examine these effects. Additionally, comparison between our delivery device and Doxil or/and Lipodox regarding release kinetics, antitumor effect and side effects will be more relevant with the requirement of this medical device.

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5. Conclusions

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In conclusion, we show here that subcutaneous implantation of DESCLAYMR_DOX can provide significant growth inhibition of human breast cancer xenografts, and can decelerate multi-organ metastasis formation. This device does not cause such adverse side effects as skin ulceration and cardiotoxicity compared with subcutaneous injection of the same dose of doxorubicin. Therefore, the DESCLAYMR_DOX implantation can be a potential treatment device for patients with bone metastases who undergo tumor-debulking surgeries. The scaffold can be tailored to fit into the void after bone tumor resection to support the structure, to sustainably release small amounts of anticancer drug to eliminate the remaining tumor cells and to avoid systemic toxicity affiliated with doses of chemotherapy.

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Acknowledgments

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We thank Vibeke Skovhus Nielsen, and the Pathology Department of the Aarhus University Hospital for expert technical assistance and conducting study on histology. The authors also express their appreciation to Linda Nygaard for her help in editing. This study is supported by the Velux Foundation, the Aarhus Spine Research Foundation and the Lundbeck Foundation Nanomedicine Center for Individualized Management of Tissue Damage and Regeneration (LUNA). This paper was presented at the 60th Annual Meeting of the Orthopaedic Research Society, New Orleans, USA, 2014.

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Appendix A. Figures with essential color discrimination

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Certain figures in this article, particularly Figs. 1 and 4–7, are difficult to interpret in black and white. The full color images can be found in the on-line version, at http://dx.doi.org/10.1016/j.actbio.2015.02.004.

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