3D printed biodegradable implants as an individualized drug delivery system for local chemotherapy of osteosarcoma

Materials and Design 186 (2020) 108336

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3D printed biodegradable implants as an individualized drug delivery system for local chemotherapy of osteosarcoma Yonghui Wang a,1, Liang Sun b,1, Zhigang Mei c, Fazhou Zhang c, Meifang He d, Cameron Fletcher e, Fenglong Wang f, Jingjing Yang f, Dongbin Bi b, Yanyan Jiang f,⁎, Ping Liu g,⁎ a

Departments of Orthopaedics, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan, 250021, China Department of Urology, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China Guangzhou Sihe Biotechnology Co., Ltd., 510530, China d Laboratory of General Surgery, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510080, China e School of Chemical Engineering, University of New South Wales, Sydney, NSW 2052, Australia f Key Laboratory for Liquid-Solid Structural Evolution and Processing of Materials, Ministry of Education, Shandong University, Jinan 250061, China g Department of Pharmacy, Shandong Provincial Hospital Affiliated to Shandong First Medical University, Jinan 250021, China b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• 3D printed PLLA drug carriers could realize the possibility of personalized local chemotherapy in the treatment of osteosarcoma. • 3D rapid prototypes suggested that the spherical implant should be more favorable for the anticancer drug delivery. • As a local chemotherapy, this novel 3D printed drug administration system has many other advantages.

a r t i c l e

i n f o

Article history: Received 16 August 2019 Received in revised form 5 November 2019 Accepted 6 November 2019 Available online 18 November 2019 Keywords: 3D printing Drug delivery implant Individualized treatment Controlled release Local chemotherapy Osteosarcoma

a b s t r a c t A 3D printing technique has been developed which enables the treatment of osteosarcoma via personalized local chemotherapy. A series of in vivo trials were conducted which closely mimicked real clinical chemotherapeutic conditions. We have shown that 3D printed poly L-lactic acid (PLLA) implants are exceptional carriers for anticancer drugs and can be designed with finely tuned physical morphologies and controllable micropore structures. Their favorable biodegradability, in vitro cytotoxicity, in vitro blood compatibility, in vivo subacute toxicity, and in vivo sensitization tests have confirmed their biocompatibility and pharmaceutical properties. Furthermore, we have demonstrated that local chemotherapy with the assistance of the as-prepared PLLA implant exhibits an anti-osteosarcoma efficacy superior to traditional chemotherapy through a series of in vivo antiosteosarcoma tests according to clinical protocols. The proposed 3D printed drug delivery system can simultaneously realize individual local chemotherapy, multi-drug delivery, long-term sustainable drug release, and non-reoperation in osteosarcoma treatment. Our studies enable the utilization of the 3D printing technique in the treatment of osteosarcomas and guide future clinical trials. The established techniques and principles can also be adapted to the local chemotherapy of other tumors.

⁎ Corresponding authors. E-mail addresses: [email protected] (Y. Jiang), [email protected] (P. Liu). 1 These authors have contributed equally to this study.

https://doi.org/10.1016/j.matdes.2019.108336 0264-1275/© 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

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Y. Wang et al. / Materials and Design 186 (2020) 108336

© 2019 Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Osteosarcoma (also called osteogenic sarcoma) is a highly aggressive form of musculoskeletal neoplasm [1]. The histologic hallmark of this cancer is the formation of malignant osteoid, and it is generally believed to originate from primitive mesenchymal bone-forming cells [2,3]. In other words, the cancer cells in these tumors resemble early forms of bone cells that normally promote osteogenesis; however, the bone tissue in osteosarcoma is not as strong as that of normal bones. Various subtypes of osteosarcoma have been recognized, and the identification of the tumor is determined by its dominant component. The community impact of osteosarcoma is frequently under-estimated. Classic osteosarcoma is a rare malignant tumor that accounts for only 0.2% of all malignant tumors and has an estimated annual incidence of 3 cases per million population [2]. However, the prognosis is extremely poor, and there is a high mortality rate and disability rate. It is the commonest malignant bone cancer among children and adolescents younger than 24 years [4] and occurs most frequently during pubertal growth spurts. Similar to the treatment of other malignant tumors, chemotherapy, radiotherapy, and surgery are the main treatment modalities currently available for osteosarcoma [3]. The most common modality for treating osteosarcoma in children involves a three-tiered approach. Firstly, chemotherapy is used to shrink the cancer by killing cancer cells via the use of medical drugs. Surgery is then required to physically remove any remaining cancerous cells and tumors before a further round of postoperative chemotherapy designed to eliminate any residual cancer cells and reduce the likelihood of the cancer returning. Long-term outcomes for patients with high-grade osteosarcoma have improved with the addition of systemic chemotherapy. Compared with 2-year overall survival rates of 15–20% following surgical resection and/or radiotherapy [1], survival rates of 60–70% can be achieved with effective chemotherapy for non-metastatic osteosarcoma [5,6]. Unfortunately, there are still many problems with the use of chemotherapy [7,8] and current treatment costs for osteosarcomas are very high [9]. These therapies are invasive, painful for patients, and are nonspecific to cancer cells. High doses of systemic chemotherapy are required to achieve sufficient localized drug concentration at the site of the lesion. This often leads to severe and undesirable adverse effects such as myelosuppression, liver and kidney dysfunction, and central nervous system disturbances that can be fatal [10]. At the very least, the patient's quality of life is significantly reduced. Furthermore, there are many osteosarcomas that are chemoresistant and radioresistant [11]. Nevertheless, it is undeniable that chemotherapy has still attracted much attention due to its potential to simplify the administration process and reduce the relative damage to tissue. To overcome both the dose-limiting adverse effects of conventional chemotherapeutic agents and the therapeutic failure incurred from drug resistance in osteosarcoma, enormous research efforts have been devoted to improving the efficacy of chemotherapy. Drug delivery systems, which are able to retain the original pharmacological action of the loaded anticancer drugs for extended periods of time while also allowing an “on-demand dose” have been considered to be extremely valuable tools in modern medicine [12]. As one of the typical modalities, nanoparticles frequently act as drug carriers for anticancer therapeutics [13,14]. The use of nanoparticle delivery systems improves outcomes across a range of different aspects such as improvement of drug solubility, enhanced cellular uptake, and bypassing multi-drug resistance. In addition, their

distribution can easily be visualized using a combination of techniques [15,16]. The nanoparticles can guide the drug to the cancer site via enhanced permeability and retention mechanisms or via cell targeting peptides or antibodies [17]. Despite the large amount of research in this area, a suitable and effective way of delivering drugs to osteosarcoma lesions has proven to be difficult for a number of reasons, such as lack of biomarker and numerous histological subtypes. Implantable drug delivery technology appeals to researchers in the areas of osteosarcoma treatment due to its advantages over conventional methods such as oral or parenteral dosage form. Such technology allows the sustained release of anticancer drugs over a period of weeks to months and sometimes even years [18]. The implantable system also acts as a means of local drug administration which results in high drug concentrations at the site of interest while reducing systemic drug exposure and thus minimizing unwanted adverse effects [19]. In the 1980s, Klemm invented the gentamicin bead chain, which constituted the first clinical application of local implantable drug carriers [20,21]. Inspired by this work, researchers have invented drug-loaded implants for local chemotherapy of tumors. Fournier et al. have found that the implant can inhibit the growth of tumor cells within 0.5–5 cm in the vicinity of the implant [22]. The drug-loaded implants combine active drugs with a biocompatible carrier and slowly release the drug after being placed in the body, thereby achieving local treatment. Increasingly more researchers and clinicians favor local chemotherapy due to its high local drug concentration, low systemic blood drug concentration, fewer adverse effects, and desirable anti-tumor efficacy. However, traditional implantable materials can only carry single drugs. The demand for secondary surgery and the inability to personalize treatment also limits its clinical application. In order to fabricate desirable implantable drug delivery devices for the treatment of osteosarcoma, biocompatibility, biodegradability, and bioactivity are all critical elements that need to be considered [23–25]. To overcome these barriers in individualized local chemotherapy, 3-dimensional (3D) printing (3DP) techniques and their products have been increasingly attracting attention [26,27]. The bespoke manufacturing of 3D-printed structures is able to provide solutions to these problems in the form of personalized anti-osteosarcoma drug delivery system. The high degree of flexibility and controllability of the 3DP technique enables the preparation of complex forms of tailored dosages with different release profiles and enhances the precision of personalized therapy [28]. Moreover, 3DP technology is getting cheaper by the day. Currently, 3DP technologies are mainly used to provide templates in order to achieve precise osteosarcoma resection [29] or fabricate high-strength bioscaffolds for bone defect regeneration [30]. To the best of our knowledge, 3DP technology has rarely been used to administer local chemotherapy in osteosarcoma treatment. In this study, for the first time, we combined individualized chemotherapy with local treatment to treat osteosarcoma. We prepared the poly L-lactic acid (PLLA) drug delivery implants with the desired structure via a 3DP technique and tested their physical parameters, biodegradability, and pharmaceutical properties (biocompatibility, cytotoxicity, sensitization, etc.). In vivo anti-osteosarcoma efficacy studies of drug release properties, general sample observation, microcomputed tomography (CT) and immunohistochemical examination were also performed. The proposed approaches may pave the way for taking advantage of such 3DP techniques to achieve individualized treatment of osteosarcoma in clinical practice.

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2. Methods 2.1. Materials Unless otherwise specified, all chemicals were of reagent grade. PLLA (molecular weight ≃60,000; Sigma-Aldrich), cisplatin (≥98% by high performance liquid chromatography (HPLC); Sigma-Aldrich), ifosfamide (≥98%; Sigma-Aldrich), methotrexate (Sigma-Aldrich), doxorubicin (Sigma-Aldrich), dichloromethane (DCM) (98%; Ajax), diethyl dimethyl sulfoxide (98%; Ajax), sodium phosphate dibasic (98%; Sigma-Aldrich), sodium phosphate monobasic (N99%; Sigma-Aldrich), and sodium chloride (Univar, reagent) were used as received. Male Sprague-Dawley (SD) rats (Southern Medical University, Guangzhou, China) weighing 275–300 g were maintained on a 12 h light/dark cycle with food and water available ad libitum. All experiments were performed in accordance with the policies and recommendations of Southern Medical University. 2.2. Fabrication of 3D printed biodegradable implants and loading of anticancer drugs Two different shapes of implants (cylindrical and spherical) were designed using the Mimics 10.0 program (Materialise NV, Leuven, Belgium) according to human anatomy and biomechanics. A stereolithography strategy was used to print the implantable 3D rapid prototypes. PLLA implants, with the sizes shown in Table S1, were printed using the 3D printer Zcorp Zprinter 650 (Z Corporation, South Carolina, USA). The thickness of the building layer was 0.05 mm, with a print resolution of 0.01 mm. The rapid prototypes were obtained after 2 h lyophilization. Based on the conventional clinical osteosarcoma postoperative chemotherapy regimen, doxorubicin (45 mg/m2/d), ifosfamide (2 g/m2/d), methotrexate (10 g/m2/d), and cisplatin (120 mg/m2/d) were accurately weighed and dissolved in a small amount of DCM. The rapid prototypes were soaked in the mixture and the DCM volatilized after exposure to the air. After further drying in a desiccator, the drug loading capacity of these rapid prototypes was measured by weighing the prototypes before and after drug loading and calculated using the following equation: Drug loading efficiency ð%Þ ¼

ðweight of prototypes after drug loading–weight of prototypes before drug loadingÞ  100 weight of prototypes before drug loading

2.3. In vitro cytotoxicity test of the spherical implants without drug loading Human osteosarcoma U2OS cells were cultured in tissue culture flasks with Dulbecco's modified eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C with 5% carbon dioxide. After reaching confluence, cells were collected from the flasks with trypsin/ethylenediaminetetraacetic acid treatment. The cell suspension was then seeded into a 96-well cell culture plate at a cell density of 5.0 × 104 cells/mL and 100 μL/well. After incubation for 1 day, the cells in the plate were subsequently used for a cytotoxicity test. This involved sterilizing the leach liquor of the rapid prototype by ultraviolet irradiation for 20 min and serially diluting the solution by half with sterile water. At the same time fresh DMEM was used as a negative control and phenol solution (64 g/L) was used as a positive control. The old medium in the cell culture plate was discarded and replaced with 100 μL of fresh twice-concentrated DMEM medium. The cells were incubated for another 48 h and then fixed by the addition of cold trichloroacetic acid. The culture medium was discarded and 100 μL of 10% trichloroacetic acid was added to each well, followed by incubation of the plates for 30 min at 4 °C. The supernatant was discarded, and the plates washed 5 times with water and air-dried. A solution of sulforhodamine B (100 μL; 0.4% w/v) in 1% acetic acid was added to each well, and the plates incubated for 15 min at room temperature. After staining, the unbound dye was removed by washing 5 times with 1% acetic acid and air-dried. Bound stains were dissolved with 200 μL 10 mM tris buffer (pH 8.5) and absorbance was measured using a Bio-Rad BenchMark microplate reader (λ = 490 nm). The data were analyzed and plotted using GraphPad Prism version 6.0 (GraphPad Software Inc., CA, USA). 2.4. In vitro assessment of the degradation of the drug-loaded PLLA implants In vitro degradation of the drug-loaded PLLA implants was determined by weight loss measurements. Experiments were performed by placing the spherical implants in conical flasks with 100 mL phosphate-buffered saline (PBS, pH 7.4) at 37 °C [31]. The flasks were then placed in a shaking incubator and 14 samples were divided into seven groups according to seven predetermined time points (0, 2, 4, 6, 8, 10, and 12 weeks). The masses of the PLLA implants were determined at each predetermined point after drying at 40 °C in a vacuum. 2.5. In vitro hemolysis assay 0.25 mL of 2% potassium oxalate was added to 5 mL venous blood obtained from New Zealand white rabbits; 4 mL of the blood mixture was then diluted with 5 mL saline to obtain the diluted blood sample. For the treatment group, the drug-loaded PLLA implants were soaked in saline according to a ratio of 0.2 g to 5 mL and incubated at 37 °C for 30 min. The diluted blood was then added at a blood to saline ratio of 0.2 mL:10 mL, followed by incubation for 1 h at 37 °C. For the negative control, a 0.2 mL diluted blood sample was added into 10 mL saline and then incubated for 1 h. For the positive control, a 0.2 mL diluted blood sample was added to 10 mL MilliQ water and incubated for 1 h. Subsequently, all the samples were centrifuged at 2000 rpm for 5 min and the supernatants were collected. Absorbance (A) of the supernatant was measured at 545 nm. The percentage of hemolysis was calculated using the following equation:

Hemolysis ð%Þ ¼

A sample–A blank ðPBSÞ  100 A water–A blank ðPBSÞ

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2.6. Dynamic blood clotting evaluation Acid citrate dextrose (ACD) solution was prepared by diluting 1.33 g of sodium citrate, 0.47 g of citric acid, and 3 g of glucose to 100 mL with distilled water; 2.5 mL of the ACD solution was added to 10 mL whole blood to generate the ACD whole blood solution. The spherical PLLA implant (0.25 g) was placed at the center of a round bottom flask and incubated at 37 °C for 5 min. 2.5 mL of ACD whole blood was injected into the flask and incubated at 37 °C for 5 min. 2.5 mL of 0.2 M calcium chloride solution was then added to the blood and shaken for 1 min, followed by 5 min incubation at 37 °C. 50 mL of saline solution was placed into a beaker, shaken for 10 min, and the supernatant obtained. The absorbance at 540 nm (OD540) was then measured. The relative absorbance of 2.5 mL ACD whole blood in 50 mL of saline was 100. As a reference value, the blood-clotting index (BCI) was calculated using the following formula: BCI ð%Þ ¼

Is  100 Iw

where Is is the OD540 of the test group and Iw is the OD540 of the control group. 2.7. Platelet adhesion test 4.5 mL of 109 mM sodium citrate solution was added to 4.5 mL of venous blood and mix gently to generate the anti-coagulated blood. The spherical PLLA implant (0.25 g) was placed in a round bottom flask, followed immediately by the addition of 1.5 mL of the anti-coagulated blood and agitated at 3 rpm for 15 min. Blank anti-coagulated blood was set as the control group. 1 mL of each blood sample was then placed in separate 50 mL centrifuge tubes, to which 19 mL of sodium citrate solution was then added. The resulting solutions were then gently mixed at room temperature for 2 h before extracting the supernatant and counting the number of platelets in the supernatant. The platelet adhesion rate was calculated as follows: Platelet adhesion rate ð%Þ

¼

number of platelets before exposure−number of platelets after exposure  100 number of platelets before exposure

2.8. In vivo subacute toxicity test Institute of Cancer Research (ICR) rats were obtained from the Laboratory Animal Center, Southern Medical University (Guangzhou, China). The animals were housed in a well-ventilated room and kept at a constant temperature of 23 ± 2 °C, a relative humidity of 40–70%, and diurnal illumination (12 h light/dark cycles). All animals were fed with standard animal chow daily and allowed free access to drinking water. The protocol of the study was approved by the Ethical Committee of Southern Medical University. All behavioral experiments were performed by an experimenter blinded to the treatment conditions. Subacute toxicity tests were performed as follows [32]. Healthy ICR rats (275–300 g) were randomly divided into 3 groups (10 in each group, half male and half female) consisting of a saline group as a control, a 1% PLLA suspension (PLLA powder suspended in saline) group, and a 10% PLLA suspension group. The rats were exposed to the saline or PLLA suspensions by oral perfusion at a dose of 20 mL/kg once a day for 28 days. Deviations in normal behavior, coat condition, discharge, movement, and mortality of the animals from different groups were monitored daily, and body weight changes were recorded every 3 days. At the end of the 28 day experiment, all the animals were anesthetized using 10% chloral hydrate solution and blood samples were taken from the abdominal aorta. All the rats were sacrificed via cervical dislocation. Selected organs (lung, liver, heart, kidney, and spleen) of each animal were isolated, weighed, and dissected for histopathological examination. The tissues were immediately rinsed with physiological saline, fixed overnight in 4% paraformaldehyde, and then dehydrated in a graded series of ethanol and embedded in paraffin for later slicing and hematoxylin and eosin (H&E) staining. 2.9. In vivo inflammatory response test New Zealand white rabbits, purchased from the Laboratory Animal Center, Southern Medical University (Guangzhou, China), were used for this study. The rabbits were 6 months old and weighed approximately 2.5 kg. All experiments were carried out according to the guidelines for the Care and Use of Animals issued by the Ethical Committee of Southern Medical University. The animals were divided into 3 groups of two rabbits each according to the time when they were to be sacrificed (2, 6, and 12 weeks). The drug-loaded PLLA implants were subjected to low-temperature plasma sterilization prior to use. After anesthesia with pentobarbital sodium via ear vein injection, all the rabbits were shaved on the backs, disinfected with iodophor disinfectant, and placed on a surgical towel. The skin was cut longitudinally with the 5th lumbar spinous process as the center. The length of the incision was about 2 cm, and the fascia was incised. Implanting sites were selected at spots about 2.5 cm from the spine. Within each group, one was implanted with the sterilized drug-loaded PLLA implant, and the other was treated as the control. After stitching the wounds, all the animals received intraperitoneal gentamicin injection for 3 days to prevent infection. The behavior, discharge, wound condition, and mortality among the animals were monitored daily. The animals were anesthetized using 10% chloral hydrate solution and sacrificed via cervical dislocation at the predetermined time points (2, 6, and 12 weeks). The PLLA implants and the surrounding tissues were collected, fixed in a 10% formalin solution and stained with H&E for histological analysis. 2.10. Treatment of animals with drug-loaded PLLA implants SD rats (specific-pathogen-free [SPF] graded) were obtained from the Laboratory Animal Center, Southern Medical University (Guangzhou, China). Similar to the ICR rats in the subacute toxicity test, these rats were also housed in a room with a constant temperature of 23 ± 2 °C, relative humidity of 40–70% and a 12 h dark/light cycle. They were fed with standard animal chow daily and had free access to drinking water. Maintenance of

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the animals and the experimental procedures performed on them were approved by the Ethical Committee of Southern Medical University. All behavioral experiments were performed by an experimenter blinded to the treatment conditions. 2.11. In vivo osteosarcoma models and drug releasing test U2OS cells were inoculated subcutaneously into the rats via the cell suspension method. When the tumor had enlarged to 1 × 1 × 1.5 cm (about 2–4 weeks), the non-necrotic osteosarcoma tissue was removed, cut into pieces of about 0.2 × 0.2 × 0.2 cm and inoculated subcutaneously into the rats under aseptic conditions. Three generations were transmitted continuously. After tumor growth was stable, the tumor tissue was cut into pieces of 0.1 × 0.1 × 0.1 cm and inoculated under the skin on the right side of the back of the rats. According to the sacrificing time after treatment, a total of 9 groups of rats (10 in each group, half male and half female) were used. Each group of rats was intraperitoneally injected with 20 g/L sodium pentobarbital at a dose of 30 mg/kg. After anesthetizing the rats, the tumor tissue and the surrounding area were disinfected with an iodophor disinfectant, and the tumor tissue was removed. After plasma sterilization, the porous PLLA drug-loaded sphere was implanted into the wound after the tumor block was removed, and the wound was sutured layer-by-layer. Gentamicin was injected intraperitoneally for 3 days after surgery to prevent infection. At 24 h, 48 h, 96 h, 8 d, 2 w, 3 w, 4 w, 8 w, and 12 w, venous blood was collected and centrifuged (at 3000 r/min, 4 °C) for 10 min. The plasma was separated and stored at −80 °C for HPLC testing. The rats were anesthetized using 10% chloral hydrate solution and sacrificed via cervical dislocation. The tumor was then removed from the surrounding tissue and stored at −80 °C. 2.12. In vivo efficacy test of the PLLA drug-loaded implants SD rats (SPF graded) were used in the in vivo efficacy test. The tumors were generated after three rounds of in vivo passaging. Thereafter, the tumor mass was re-implanted subcutaneously in the nude mice with a size of 0.1 × 0.1 × 0.1 cm for the subsequent treatment. The tumor was established for about 2–3 weeks, and tumor formation was visible to the desired size of approximately 229 mm3. SD rats (SPF graded) bearing the aforementioned U2OS tumor tissues on the right side of the back were divided into 3 groups (6 rats in each group) according to the different treatment regimens. When the tumor volume was about 229 mm3, the rats in each group were injected intraperitoneally with 20 g/L sodium pentobarbital at a dose of 30 mg/kg. After anesthetizing the nude rats, the tumor tissue and the surrounding area were disinfected with an iodophor disinfectant, and the tumor tissue was removed. For the control group, after the tumor was resected, the wound was sutured layer by layer and gentamicin was injected intraperitoneally to prevent infection. For the normal chemotherapy group, after tumor resection, the wound was sutured layer by layer, gentamicin was injected intraperitoneally, and methotrexate, cisplatin, doxorubicin, and ifosfamide were continuously administered via intraperitoneal injection. For the PLLA drug-loaded implant treatment group, after the tumor was resected, an equal volume of the drug-loaded implant was inserted, and gentamicin was injected intraperitoneally to prevent infection. Sodium pentobarbital (20 g/L) was injected intraperitoneally at a dose of 30 mg/kg. After the mice were anesthetized, a micro-CT scan was performed at 0, 1, 4, 8, and 12 months after surgery. After the results were obtained, the Digital Imaging and Communications in Medicine standard was introduced into the repeater and reconstructed using the Mimics software. The rats were anesthetized using 10% chloral hydrate solution and sacrificed via cervical dislocation at 12 months after surgery. Separate groups of animals were sacrificed and treated afterward with mono sodium iodoacetate or saline. The legs of the animals were fixed in 10% neutral buffered formalin for 24 h and decalcified (Decalcifier 1, Surgipath; Leica Microsystems Inc., Buffalo Grove, IL, USA) for 48 h. The joints were embedded in paraffin in the frontal plane and 8–l m slices were taken at the center of the joint space. Staining with toluidine blue (0.04%) was performed, and the amount of cartilage lining the medial femorotibial joint was assessed by calculating the area using Image J software (National Institutes of Health, Bethesda, Maryland, USA). The surrounding tissues were taken and fixed in 10% formalin fixative. Markers of osteosarcoma were assayed, including Snail, MHC class I chain-related molecule A (MICA), RASSF1A, Her-2, MMP-2, MMP-9, c-Kit, and NF-κB. 2.13. Statistical analysis The measurement indices are presented as means±standard deviations. Overall comparison of the measurement indices between groups was performed via analysis of variance. Multiple comparisons of the means for each group and the model group were performed using the NewmanKeuls test. The independent t-test was used for comparisons between two groups. All statistical analyses were performed using SPSS version 20.0 (IBM, Armonk, NY, USA). Analysis items with p b .05 were considered statistically significant. 3. Results and discussion 3.1. Fabrication and characterization of the biodegradable PLLA implants Osteosarcoma is a highly malignant primary intramedullary tumor, and is the most common malignant bone tumor, accounting for about 40% of primary malignant bone tumors of the entire body [33]. The most common sites of occurrence are the metaphysical region of the distal femur and the proximal humerus, as well as the backbone [34]. Pre- and post-operative high-dose chemotherapy can improve the disease-free survival rate to 60–70% [5]; however, the adverse effects of systemic high-dose chemotherapy cannot be neglected. The implantable drug-loaded carriers used in local chemotherapy can slowly release the drug after administration, thereby reducing systemic exposure to chemotherapy drugs. Using a 3DP technique for the preparation of this type of drug-loaded implant has a broad range of advantages,

including individualized customization, the ability to load multiple drugs and control their release, rapid and precise physical production, availability of a diverse range of materials, and no direct correlation between production costs and design complexity. In this study, we fabricated the 3D printed implantable drug carriers according to the scheme shown in Fig. 1a. Mimics 10.0 software was employed to design spherical and cylindrical implants (Fig. 1b and c, respectively) according to human anatomy and biomechanics. PLLA powder was then processed into a design model using stereolithography 3DP technology. After printing, the unloaded implants were removed and freeze-dried to obtain the final products (Fig. 1d). The drugloaded implants were then prepared using a solvent evaporation method. The drug solution was prepared according to the ratios used in a standard osteosarcoma post-operative clinical chemotherapy regimen by dissolving doxorubicin (45 mg/m2/d), ifosfamide (2 g/m2/d), methotrexate (10 g/m2/d), cisplatin (120 mg/m2/d), in a small amount

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Fig. 1. Design, fabrication and characterization of the 3D printed implants. (a) Schematic illustration of the fabrication process; the cylindrical (b) and spherical (c) 3D model of the implants; (d) the configuration of the 3D printed PLLA implants; (e) the drug loading efficiency of the 3D rapid prototypes; (f)SEM image of the surface morphology of the 3D printed implants; and (g) SEM image of the inner structure of the implants.

of DCM. The drug solution was then thoroughly mixed with both types of shaped implants. DCM was then removed via evaporation by gently heating the mixture without stirring. After the solvent was completely volatilized, the implantable rapid prototypes were measured to obtain their size parameters as listed in Table S1 and weighed to calculate the drug encapsulation efficiency as shown in Fig. 1e and Table 1. The drug loading efficiency of the spherical

implant S-8 (29.8 ± 2.4%) was much higher than that of the two cylindrical ones C-9 (15.5 ± 2.4%) and C-12 (14.5 ± 2.4%), which indicates that the shape of the implant had a strong effect on the encapsulation efficiency of the 3D printed implants. Due to the increased encapsulation efficiency, we chose the spherical implants S-8 as the local drug delivery system for the subsequent investigation. Scanning electron microscopy was used to visualize the inner

Table 1 The drug loading efficiency of the 3D rapid prototypings. C-9

C-12

S-8

Before m1 (mg)

After m2 (mg)

Drug (%)

Before m1 (mg)

After m2 (mg)

Drug (%)

Before m1 (mg)

After m2 (mg)

Drug (%)

80.6 72.9 85.7 Mean ± SD

91.6 86.2 98.2

13.65 18.24 14.59 15.5 ± 2.4

256.50 249.60 262.70

293.5 291.8 294.5

14.42 16.91 12.11 14.5 ± 2.4

218.9 223.7 204.7

278.90 295.70 265.80

27.41 32.19 29.85 29.8 ± 2.4

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Fig. 2. (a) In vitro cytotoxicity evaluation of the PLLA implants. (b) The weight loss of the drug loaded PLLA implant in PBS buffer.

Table 2 In vitro hemolysis test of the drug-loaded PLLA implants. Group

Negative Positive Treatment

OD545

Percentage of hemolysis (%)

1

2

3

Mean

0.216 0.448 0.226

0.208 0.435 0.214

0.211 0.429 0.218

0.212 0.437 0.219

0.00 100.00 3.40

structure of these implants. The implants had a rough surface and a microscale porous internal morphology (Fig. 1f and g), which were conducive to cell adhesion and drug encapsulation. In addition, the porosity of the spherical implants was as high as 85.14 ± 2.16% (Table S2), conforming to the requirements for drug loading (porosity N80%), which controls the drug loading efficiency and releasing rate. 3.2. In vitro biosafety assessments of the PLLA implants Biosafety assessments are necessary for any new biomaterial or any new application of currently known biomaterials prior to clinical application. The significance of the biosafety evaluation is to predict potential hazards of biological materials or medical devices in direct contact with the human body, and to ensure their clinical compatibility [54]. Furthermore, the implantable materials are supposed to be degradable in a biological environment, such that their degradation products can participate in the normal cellular physiological and biochemical processes. It is even considered that these new materials should provide sufficient stability for the early and middle stages of various types of bone healing [55]. After performing their function, the implants are gradually degraded and absorbed by the body [35]. As degradation progresses, the stress load is gradually transferred to the healing bone.

PLLA is a valid biodegradable biomaterial with high bio-intensity [36,37], approved by the US Food and Drug Administration for certain human clinical applications [38]. We studied the cytotoxicity of the spherical PLLA implants without drug loading (Fig. 2a, S1 and Table S3). Compared with the treatment of the negative control (DMEM medium) and the positive control (phenol), we confirmed that the toxicity level of the PLLA implants was grade 0 or 1, indicating that the implants were non-cytotoxic and had good biocompatibility. We further examined the biodegradability of the drug-loaded PLLA implants in PBS. Fig. 2b shows the weight loss of the PLLA implants at all the predetermined time points, suggesting that the implants exhibited an excellent level of biodegradability [38]. The pH of the PBS decreased from 7.4 to 6.3 during the 12 week incubation period (Fig. S2), indicating that the acidic degradation of PLLA as a bone tissue engineering scaffold was less harmful. This is also in agreement with the higher degradation rate after 8 weeks because the slightly acidic environment will enhance the degradation of PLLA. No hemolysis was observed in the PLLA treatment group compared with the negative and positive control groups (Table 2). Furthermore, the BCI (Table S4) and platelet adhesion rate (Table S5) indicated that the PLLA implants would not result in blood clotting response. 3.3. In vivo biosafety assessments of the PLLA implants In addition to the aforementioned in vitro tests, we also examined the subacute toxicity and inflammatory response of the as-synthesized drug-loaded spherical PLLA implants in vivo. The animals in the test group (who received either 1% or 10% PLLA suspension) had normal body weight growth, and there was no significant difference from the control group (Fig. 3a and b). No deaths occurred among the animals throughout the study period (Table 3). All the test animals were in good health condition after PLLA suspension administration and the

Fig. 3. Body weight variation of (a) the female rats (g, Mean ± SD); and (b) the male rats (g, Mean ± SD) after PLLA suspension administration.

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Table 3 Summary of the clinical observation of the test rats after PLLA suspension administration. Symptom

Control

1% suspension

10% suspension

Female

Male

Female

Male

Female

Male

Death

0/5

0/5

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diet and autonomic activities were normal. We further investigated the subacute effect on the main organs (liver, kidney, heart, lung, and spleen) of the rats. The H&E stained light micrographs showed no clear organic damage in both treatment groups (Fig. 4). As a result of these tests, no subacute toxicity was found in the test animals. In addition to these studies, we implanted the drug-loaded PLLA spheres into

New Zealand white rabbits to observe the sensitizing effect. During the experiment, there were no symptoms such as redness, exudation or suppuration in the surgical wounds. All test animals healed in the first stage of the wound. The tissues surrounding the wounds only showed a slight inflammatory response as a result of implanting the PLLA sphere (Fig. 5). Based on the results of the systematic

Fig. 4. Histology of heart, kidney and lung in rats. Light micrographs of organs in rats were observed after 28-days oral perfusion administration, stained with H&E (100×).

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Fig. 5. Histology of tissues around the implanting site of the drug-loaded PLLA implants in rabbits stained with H&E (100×).

investigations, it is rational to conclude that our PLLA implants have a high degree of biosafety as a local chemotherapeutic agent in antiosteosarcoma treatment. 3.4. In vivo pharmacokinetic study and the efficacy of the antiosteosarcoma treatment Through this study, we successfully developed a drug-loaded implant based on the 3DP technology. Combining 3DP technology with local chemotherapy can offer many advantages. For example, a highly targeted combination of chemotherapeutic drugs can be prepared based on individual patient drug sensitivity tests and tumor pathology. The 3D printed implant allows for multi-drug loading and dispersion, whereby the concentration and speed of drug release can be precisely regulated. Most importantly, based on the specific conditions of the whole body and lesions, a selective treatment plan can be easily formulated for systemic, intramedullary, perilesional, and metastatic sites. Drug-loaded implants of different shapes and sizes can also be manufactured according to the cancer site and surgical results, to meet the individual needs of different patients. The drug release kinetics of the 3D printed drug-loaded implants were examined in animal models. The plasma samples obtained from the rats treated by local chemotherapy were assessed via HPLC after being subjected to liquid-liquid extraction in order to analyze the drug release kinetics of the four drugs including methotrexate, doxorubicin, ifosfamide and cisplatin among rats (Fig. 6a and b). The concentration of all the four drugs reached the peak value after approximately 2 weeks and the sustained release of the spherical PLLA drug carriers in the mouse body could last for N12 weeks. Moreover, by comparing Fig. 6a to b, it can be found that the drug concentration in the lesion tissue around the site of the implants is higher than that in the whole blood, which implies that there will be fewer adverse effects in the normal non-cancerous tissue. Therefore, the drug can be administered locally when the drug-loaded implant is placed in a location where there may be residual tumor cells, maintaining a high drug concentration for an extended period with fewer systemic adverse effects. In addition to this, it provides a reliable analytical method for the pharmacokinetic study of the spherical PLLA drug-loaded implants and has important guiding significance for empirical clinical treatment. To verify the therapeutic efficiency of the drug-loaded PLLA in the treatment of osteosarcoma, we established the subcutaneous osteosarcoma xenograft animal model and conducted in vivo studies by dividing the animals into three groups. After tumor resection in the control group, we sutured the wound layer by layer and intraperitoneally injected gentamicin to prevent infection. In the normal chemotherapy

group, we sutured the wound layer by layer after tumor resection and intraperitoneally injected gentamicin followed by continuous intraperitoneal injection of methotrexate, cisplatin, doxorubicin, and ifosfamide. In the PLLA implant group, an equal volume of drug-loaded implants was administered after tumor resection and gentamicin was injected intraperitoneally to prevent infection. The treatments were continued for approximately one year to mimic clinical practice. Clinical observation and immunohistochemistry were performed to evaluate the efficacy of each treatment. During the experiment, there were 3 deaths in the normal chemotherapy group and the other animals in this group suffered reduced spontaneous activity and hair loss. No abnormalities were observed in both the control group and the PLLA implants group, which further indicated that local chemotherapy with the assistance of the PLLA drug loaded implants alleviate the toxic effect caused by traditional chemotherapy. Osteosarcoma biomarkers were assessed via immunohistochemistry and illustrated the anti-osteosarcoma efficacy

Fig. 6. (a) The drug concentration of the lesion tissue around the implanting site. (b) The drug concentration in the systematic blood. Micro-CT images of the thoracic abdominal transverse section of the rats with local chemotherapy treatment. The concentration of methotrexate, doxorubicin, ifosfamide and cisplatin in the rabbit.

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of the drug-loaded PLLA sphere (Figs. 7 and 8). Our results were consistent with the recent literature [39,40], suggesting that the biomarkers, such as matrix metalloproteinase-2, NF-κb, MICA and the like, are involved in the development, progression, and invasion of osteosarcoma.

It is worth noting that, according to the immunohistochemistry results, especially the mean density analysis (Fig. 8), there were lower levels of expression of almost all the mentioned biomarkers in the experiment group treated with the PLLA drug-loaded implants. These results

Fig. 7. The immuno-histochemistry examination of the tissues around the tumor site of different treatments after 12 months.

Y. Wang et al. / Materials and Design 186 (2020) 108336

Fig. 8. Mean density analysis of the immuno-histochemistry examination of the tissues around the tumor site of different treatments after 12 months. * p b .05, **p b .01, n = 6.

strongly imply that local chemotherapy with the assistance of the 3D printed drug-loaded PLLA sphere is much more effective than traditional chemotherapy. Clinical observation, micro-CT scans, and immunohistochemistry were performed to evaluate the efficacy of each treatment. During the experiment, there were 3 deaths in the normal chemotherapy group and the other animals in this group had reduced spontaneous activity and hair loss. No abnormalities were observed in both the control group and the PLLA implants group, which further indicated that local chemotherapy with the assistance of the PLLA drug loaded implants could evidently alleviate the toxic effects of traditional chemotherapy. During the entire postoperative period (1–12 months), the abdominal tissues or organs in the CT images showed clear boundaries and no obvious shadows, indicating that there was no tumor recurrence. This

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implied that PLLA drug loading implantation could effectively inhibit the growth and recurrence of osteosarcoma (Fig. 9). In the 1980s, rapid prototyping techniques first appeared in the United States. The main characteristic of this technique was its unique manufacturing method. To complete the manufacture of the product, materials are piled up and combined layer by layer, of which the whole process is controlled by a computer. The rapid prototyping techniques do not depend on molds, which is different from the traditional manufacturing techniques. Since then, various rapid prototyping techniques had been developed. It is capable of manufacturing products with complex shapes and intricate internal structures, which has a great application potential in the medical field. Tissue engineering scaffolds with unique structures could be prepared by using 3DP machines [41]. It also enabled to manufacture individualized bone fracture models as the preoperative reference for doctors according to the 3D CT data, and fabricate individualized implants for fixing bone defects [42]. The local slow release implant enables to provide long term and stable drug release in the lesion. In contrast to oral drug administration, it increases the topical drug concentration while maintains the blood drug concentration at a relatively low level. Therefore, the drug therapeutic efficacy is enhanced while the systemic adverse effects are alleviated. Recently, local drug delivery systems have been mainly utilized to treat tumors and chronic infectious diseases [43,44]. To manufacture drug delivery devices with the ability of releasing drugs smoothly in a release rate at or close to zero, various drug loading devices have been explored, such as doughnut shaped tablets, multilayer tablets and micro particles, etc. [45–47].

Fig. 9. Micro-CT images of the thoracic abdominal transverse section of the mice with local chemotherapy treatment. (a) before surgery, (b)0 month, (c)1 months, (d)4 months, (e) 8 months, and (f)12 months after the implanting treatment.

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Since the 3DP technique was first introduced, lots of attention has been attracted to its application [48–50]. The 3D structure models designed by computer are fabricated using 3DP machine by binding powders layer by layer or by binding liquid. It has great advantages in fabricating drug loading implants with complicated internal structures [51–53]. Model drugs can be evenly dissolved in the binding liquid and precisely injected on the powder layer by the printing head. Since the drug loading ability of the implant is limited, the effective therapeutic concentration of the model drug should be low. This novel 3D printed drug administration system, as a local chemotherapy, has some other advantages. The local drug concentration is extremely high, greatly improving the drug efficacy. The drug can directly act on the local tumor site to kill the remaining cancer cells, resulting in the alleviation of the toxic effect caused by systemic chemotherapy. Furthermore, the topical drug release can be sustained and the drug action duration can be prolonged. 4. Conclusions In summary, we have developed a novel strategy using 3D printed PLLA drug carriers that may realize the potential of personalized local chemotherapy in the treatment of osteosarcoma. Systematic examination of the physical properties of the various 3D rapid prototypes suggested that the spherical implant should be more favorable to the anticancer drug delivery device for individual dosage and therapy, multi-drug delivery, and long-term sustainable drug release. More importantly, the PLLA implants are biodegradable with high biocompatibility and biosafety. Our results demonstrated that there was no redness, exudation, suppuration, or other infections in the wounds of the test animals. As local chemotherapy, this novel 3D printed drug administration system has many other advantages. The local drug concentration was extremely high enabling the drug efficacy to be greatly improved. It directly acts on the local tumor site to kill the remaining cancer cells so that the toxic effects caused by systemic chemotherapy can be alleviated. Furthermore, sustained release of topical drugs and the prolongation of the duration of drug action is achievable. The technique established in this study could have a profound influence on clinical trials providing researchers with the ability to fabricate desired geometries to achieve variable drug release kinetics, ease individual pharmacotherapy for patients, provide bone support during treatment, and lower the cost of personalized treatment. The technique described herein thus represents a potential universal platform for antiosteosarcoma therapy. CRediT authorship contribution statement Yonghui Wang: Data curation, Formal analysis.Liang Sun: Investigation. Zhigang Mei: Methodology. Fazhou Zhang: Methodology. Meifang He: Validation, Methodology. Cameron Fletcher: Writing - review & editing. Fenglong Wang: Writing - original draft. Jingjing Yang: Writing - original draft. Dongbin Bi: Writing - original draft, Resources. Yanyan Jiang: Conceptualization, Writing - review & editing, Supervision. Ping Liu: Conceptualization, Writing - review & editing, Supervision. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to acknowledge the support from the National Natural Science Foundation of China (Grant No. 81874034, 5167111, 81271663 and 31471146), (No. 2019GSF108002,

2016GSF201156), the Medical and Health Science and Technology Development project of Shandong Province (No. 2017WS198), the Science and Technology Development Plan of Shandong Province (No. 2014GSF118092), the Medical Health Science and Technology Innovation Plan of Jinan (No. 201805038) and the Fundamental Research Fund of Shandong University (Grant No. 2018CJ047). This work is also supported by Qilu Young Scholar Program of Shandong University. References [1] N. Marina, M. Gebhardt, L. Teot, R. Gorlick, Biology and therapeutic advances for pediatric osteosarcoma, Oncologist 9 (2004) 422–441. [2] P. Picci, Osteosarcoma (osteogenic sarcoma), Orphanet J. Rare Dis. 2 (2007) 6. [3] T. Heare, M.A. Hensley, S. Dell’Orfano, Bone tumors: osteosarcoma and Ewing’s sarcoma, Curr. Opin. Pediatr. 21 (2009) 365–372. [4] M.A.M. Guillon, P.M.J. Mary, L. Brugière, P. Marec-Bérard, H.D. Pacquement, C. Schmitt, J.-M. Guinebretière, M.-D.P. 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