Elsevier

Acta Biomaterialia

Volume 118, December 2020, Pages 69-82
Acta Biomaterialia

A 3D-printed biomaterials-based platform to advance established therapy avenues against primary bone cancers

https://doi.org/10.1016/j.actbio.2020.10.006Get rights and content

Abstract

In this study we developed and validated a 3D-printed drug delivery system (3DPDDS) to 1) improve local treatment efficacy of commonly applied chemotherapeutic agents in bone cancers to ultimately decrease their systemic side effects and 2) explore its concomitant diagnostic potential. Thus, we locally applied 3D-printed medical-grade polycaprolactone (mPCL) scaffolds loaded with Doxorubicin (DOX) and measured its effect in a humanized primary bone cancer model. A bioengineered species-sensitive orthotopic humanized bone niche was established at the femur of NOD-SCID IL2Rγnull (NSG) mice. After 6 weeks of in vivo maturation into a humanized ossicle, Luc-SAOS-2 cells were injected orthotopically to induce local growth of osteosarcoma (OS). After 16 weeks of OS development, a biopsy-like defect was created within the tumor tissue to locally implant the 3DPDDS with 3 different DOX loading doses into the defect zone. Histo- and morphological analysis demonstrated a typical invasive OS growth pattern inside a functionally intact humanized ossicle as well as metastatic spread to the murine lung parenchyma. Analysis of the 3DPDDS revealed the implants ability to inhibit tumor infiltration and showed local tumor cell death adjacent to the scaffolds without any systemic side effects. Together these results indicate a therapeutic and diagnostic capacity of 3DPDDS in an orthotopic humanized OS tumor model.

Introduction

The overall 5-year survival rate of patients diagnosed with Osteosarcoma (OS), the most common type of primary bone cancer, has stagnated at approximately 60% since the 1960s [1]. Infiltrating tumor growth, local reoccurrence and early pulmonary metastatic spread are still the leading causes of mortality. At the time of clinical manifestation, metastatic spread has occurred in about 20% of OS patients with only 1 out of 4 of these patients (24-26.7%) surviving longer than 10 years [2], [3], [4]. For recurrent OS the 5-year survival rate drops to less than 25%, resulting in an overall poor prognosis for patients diagnosed with this disease [4].

Besides removal of the primary and metastatic lesions, Doxorubicin (DOX), Cisplatin, Methotrexate and Ifosfamide are currently the predominant chemotherapeutic treatment agents, to prevent local relapse or metastatic spread [5]. However, since the implementation of neoadjuvant and adjuvant chemotherapy in combination with surgical resection during the 1960s and 1970s, the therapeutic regime for OS has remained largely unchanged. In order to further improve the outcome of OS patients and overcome the stagnating 5-year survival rate, new drugs or innovative treatment strategies are required.

Two major challenges in the development of new treatment options for cancer patients are (1) the low translatability and reproducibility of promising preclinical results into the clinic and (2) the plethora of side effects of cytotoxic anti-tumor agents / chemotherapeutics.

Xenograft mouse models are considered the gold standard for preclinical cancer research. However, there is a failure rate of over 80% in efforts to translate preclinical results from animal studies to the human physiology [6]. In this context it is well known that despite the phylogenetic similarity of humans and mice, minor interspecific differences in DNA protein coding sequences can have a large impact on the peptide mass [7]. As for collagen, the most abundant extracellular matrix (ECM) protein in bone, there is a 89% genetic match between mice and humans with 37 changes in amino acid composition after translation [8]. Changes like this can not only alter ECM composition, but also influence signaling pathways and direct reciprocal cellular interactions [9] which in turn are known to be play a crucial role in tumorigenesis [10]. To overcome this translational obstacle, humanized mouse models aim to create a human tissue environment within a murine host to account for essential species-specific crosstalk between human cells and their local habitat. This way, a human disease can be replicated more faithfully to ultimately study the human and not the animals disease pathology. Recent chimeric liver mouse models for example show great potential to significantly improve preclinical drug testing and thus promises great utility for future pharmacological studies [11, 12].

Another major challenge in modern tumor therapy is the non-specificity of commonly used drugs, resulting in debilitating side effects which have a significant impact on patients’ quality of life [13]. Even though employed to treat the underlying disease, surgery as well as chemotherapy cause significant injury to healthy tissues and organs. While resection of the tumor can be focused on a predefined area, administration of common chemotherapeutic drugs per os (orally) or via an intravenous route, exposes tissues even at distant sites to toxic amounts of the respective agent. For anthracyclines like DOX, cardio-, hepato- and nephrotoxic effects are well known and described [13], [14], [15]. Ultimately the accumulation of DOX in certain tissues can lead to fatal organ dysfunctions such as congestive heart failure [16]. Hence, the safe cumulative dose of DOX that can be administered has been limited to 400-500 mg/m2 [17]. However, to achieve a therapeutic effect against tumor cells surrounded by calcified tissue, increased dosages are necessary [18]. Yet, advanced treatment strategies such as attaching DOX to liposomes - which significantly reduced cardiotoxicity- [19], as well as novel multimodal treatment regimes, were not able to significantly improve the relatively poor outcome of OS patients.

In this regard, new treatment modalities aim to increase local bioavailability of cytotoxic drugs and reduce exposure to healthy tissues by either specifically guiding the drug to the tumor site or locally implanting a drug delivery system [20]. The former method relies on specific targeting of biological characteristics (epitopes), to manipulate or interfere with cell signaling, proliferation, angiogenesis or apoptosis pathways [21]. However, promising targeting drugs such as imatinib (a tyrosine kinase inhibitor), still trigger various side effects and failed to show efficacy when tested on patients with OS [22], underlining the challenge to develop effective targeted therapies. Reviewing targeted therapies for OS, Sampson et al. concluded that: “Many biological agents demonstrating anti-tumor responses in preclinical and early-phase clinical testing have failed to reach response thresholds to justify randomized trials with large numbers of patients” [21]. In contrast, implantation of a drug delivery system does not require a molecular target to increases local bioavailability, as such a device can be directly placed adjacent to or inside the tumor tissue. For OS treatment recent studies have used thermo-sensitive hydrogels [23] or 3D-dimensional (3D) scaffolds [24] loaded with anti-tumor agents to achieve a sustained drugs release after local implantation. In particular with the recent advancements of 3D-printing techniques we now have the unique opportunity to explore new local treatment solutions by designing highly customizable and patient-specific drug delivery systems - “addressing unmet clinical needs” [25, 26]. Utilizing this 21st century technology, carrier materials can be individually manufactured in any required shape and with tunable release profiles to dispense a predefined drug volume with accurate spatiotemporal control [27, 28].

Besides being of therapeutic value as a delivery system for chemotherapeutic drugs, such a device could be designed to additionally evaluate dosing requirements or individual tumor characteristics such as invasiveness and chemoresistance. This patient-specific information could guide clinicians in their therapeutic decisions e.g. to avoid low drug efficacy while concurrently causing detrimental off-target side effects. However, to be utilized as a diagnostic tool that could have an impact on treatment plans, such a device would need to be applied early in the course of the disease. Here, the time from biopsy to treatment initiation creates a unique opportunity to insert such a device into the biopsy defect zone that could help to advance currently established therapeutic avenues.

The biopsy procedure is the key step in clinical practice which is necessary to establish the diagnosis of OS. If clinical evaluation of the patient indicates the presence of a primary bone cancer, the clinician will take several biopsies of the lesion while a pathologist will then classify the cancerous cells (grading). After biopsy it is not uncommon that patients are required to wait 2-3 weeks until diagnosis and an additional 3 weeks before actual treatment can be commenced [29]. This “therapeutic gap” offers the opportunity to explore new methods to improve and direct outcome of a potentially lethal disease. Here, the implantation of a local therapeutic and/or diagnostic scaffold into the defect zone at the timepoint of biopsy can bridge the time until final pathology results are available. In this way a local drug delivery system implanted into the tumor could not only be a solution to overcome the general problem of low local bioavailability and reduce DOX exposure to distant tissues, but also be of diagnostic value.

We thus hypothesized that the local implantation of a 3DPDDS / 3D-printed mPCL-scaffold loaded with DOX would have a therapeutical impact on the surrounding tumor tissue without causing any significant side effects on distant organs. Concomitantly we predicted that immunohistochemical analysis of tissues grown inside the scaffold architecture provides important information about the local tumor pathology.

Section snippets

Scaffold design & manufacturing, drug loading and cell culture

Tissue engineering a humanized bone organ model using 3D-printed mPCL-scaffolds has been previously described and characterized by our group [30]. In short, by employing melt-electrowriting technology [31], a class of additive manufacturing technology, three different types of tubular scaffolds were manufactured for this study (suppl. Table 1).

  • (1)

    Scaffolds manufactured to host human osteoblast progenitor cells (hOBs) were surface-activated with NaOH, coated with Calcium phosphate (CaP) and

Drug loading efficacy and release kinetics of Doxorubicin laden scaffolds

DOX loaded onto mPCL scaffolds resulted in a gradually increased red stain of the scaffold fibres from low dose (LD) to high dose (HD) (Fig. 1A). SEM imaging depicted a smooth morphology of scaffold fibres indicating a homogenous absorption of DOX. Spectrophotometry revealed a drug loading efficacy (total drug released against DOX amount loaded) of 90% ± 1% for LD, 94% ± 2% for MD and 95.1% ± 0.5% for HD DOX scaffolds. The in vitro release assay showed that within 48 hours approximately 72%,

Discussion

This study was designed to develop a new treatment strategy that could bridge the “therapeutic gap” by: implanting a biodegradable, tuneable 3D-printed mPCL-scaffold loaded with DOX as a local drug delivery system and diagnostic tool for the treatment of primary bone cancers. First, we established an orthotopic humanized niche for the development of an OS lesion via bone tissue engineering techniques. In a next step, this model was used as a platform to assess the local application of DOX

Conclusion

In summary, we utilized a humanized OS model to demonstrate the potential of 3DPDDS as a therapeutic and diagnostic tool to ultimately bridge the therapeutic gap of current treatment regimens for OS. The presented work could serve as platform to investigate how a locally implanted 3DPDDS can influence tumorigenesis in a humanized environment, explore the tumor-associated immune response and pave the way for innovative therapeutic and diagnostic approaches. Overall, 3D printing techniques are a

Conflict of Interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

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.

Acknowledgments

The authors would like to acknowledge the scientific and technical assistance of the Translational Research Institute Preclinical Imaging Facility and Biological Resources Facility. Baxter Healthcare Australia kindly provided Fibrin Glue (TISSEEL Fibrin Sealant). The following funding existed during conduction of this study: National Health and Medical Research Council of Australia (Project Grant 1082313) and Australian Research Council (ARC ITCC 160100026).

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