3D bone models to study the complex physical and cellular interactions between tumor and the bone microenvironment.

As the complexity of interactions between tumor and its microenvironment has become more evident, a critical need to engineer in vitro models that veritably recapitulate the 3D microenvironment and relevant cell populations has arisen. This need has caused many groups to move away from the traditional 2D, tissue culture plastic paradigms in favor of 3D models with materials that more closely replicate the in vivo milieu. Creating these 3D models remains a difficult endeavor for hard and soft tissues alike as the selection of materials, fabrication processes, and optimal conditions for supporting multiple cell populations makes model development a nontrivial task. Bone tissue in particular is uniquely difficult to model in part because of the limited availability of materials that can accurately capture bone rigidity and architecture, and also due to the dependence of both bone and tumor cell behavior on mechanical signaling. Additionally, the bone is a complex cellular microenvironment with multiple cell types present, including relatively immature, pluripotent cells in the bone marrow. This prospect will focus on the current 3D models in development to more accurately replicate the bone microenvironment, which will help facilitate improved understanding of bone turnover, tumor-bone interactions, and drug response. These studies have demonstrated the importance of accurately modelling the bone microenvironment in order to fully understand signaling and drug response, and the significant effects that model properties such as architecture, rigidity, and dynamic mechanical factors have on tumor and bone cell response.

Engineering 3D Models of Tumors and Bone to Understand Tumor-Induced Bone Disease and Improve Treatments.

PURPOSE OF REVIEW: Bone is a structurally unique microenvironment that presents many challenges for the development of 3D models for studying bone physiology and diseases, including cancer. As researchers continue to investigate the interactions within the bone microenvironment, the development of 3D models of bone has become critical. RECENT FINDINGS: 3D models have been developed that replicate some properties of bone, but have not fully reproduced the complex structural and cellular composition of the bone microenvironment. This review will discuss 3D models including polyurethane, silk, and collagen scaffolds that have been developed to study tumor-induced bone disease. In addition, we discuss 3D printing techniques used to better replicate the structure of bone. 3D models that better replicate the bone microenvironment will help researchers better understand the dynamic interactions between tumors and the bone microenvironment, ultimately leading to better models for testing therapeutics and predicting patient outcomes.

A Perfusion Bioreactor Model of Tumor-Induced Bone Disease Using Human Cells.

Advanced solid tumors often metastasize to bone. Once established in bone, these tumors can induce bone destruction resulting in decreased quality of life and increased mortality. Neither 2D in vitro models nor 3D animal models sufficiently recapitulate the human bone-tumor microenvironment needed to fully understand the complexities of bone metastasis, highlighting the need for new models. A 3D in vitro humanized model of tumor-induced bone disease was developed by dynamically culturing human osteoblast, osteoclast, and metastatic cancer cells together within tissue-engineered bone constructs. Cell-mediated resorption can be observed by micro-computed tomography and can be quantified by change in mass. Taken together, these data can be used to investigate whether the metastatic cancer cells included in the model have the potential to drive osteoclastogenesis and cell-mediated resorption in vitro. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Fabricating bone-like scaffolds Basic Protocol 2: Preparing cells for the humanized model of TIBD Basic Protocol 3: Crafting a 3D in vitro humanized model of TIBD.

Tuning Ligand Density To Optimize Pharmacokinetics of Targeted Nanoparticles for Dual Protection against Tumor-Induced Bone Destruction.

Breast cancer patients are at high risk for bone metastasis. Metastatic bone disease is a major clinical problem that leads to a reduction in mobility, increased risk of pathologic fracture, severe bone pain, and other skeletal-related events. The transcription factor Gli2 drives expression of parathyroid hormone-related protein (PTHrP), which activates osteoclast-mediated bone destruction, and previous studies showed that Gli2 genetic repression in bone-metastatic tumor cells significantly reduces tumor-induced bone destruction. Small molecule inhibitors of Gli2 have been identified; however, the lipophilicity and poor pharmacokinetic profile of these compounds have precluded their success in vivo. In this study, we designed a bone-targeted nanoparticle (BTNP) comprising an amphiphilic diblock copolymer of poly[(propylene sulfide)-block-(alendronate acrylamide-co-N,N-dimethylacrylamide)] [PPS-b-P(Aln-co-DMA)] to encapsulate and preferentially deliver a small molecule Gli2 inhibitor, GANT58, to bone-associated tumors. The mol % of the bisphosphonate Aln in the hydrophilic polymer block was varied in order to optimize BTNP targeting to tumor-associated bone by a combination of nonspecific tumor accumulation (presumably through the enhanced permeation and retention effect) and active bone binding. Although 100% functionalization with Aln created BTNPs with strong bone binding, these BTNPs had highly negative zeta-potential, resulting in shorter circulation time, greater liver uptake, and less distribution to metastatic tumors in bone. However, 10 mol % of Aln in the hydrophilic block generated a formulation with a favorable balance of systemic pharmacokinetics and bone binding, providing the highest bone/liver biodistribution ratio among formulations tested. In an intracardiac tumor cell injection model of breast cancer bone metastasis, treatment with the lead candidate GANT58-BTNP formulation decreased tumor-associated bone lesion area 3-fold and increased bone volume fraction in the tibiae of the mice 2.5-fold. Aln conferred bone targeting to the GANT58-BTNPs, which increased GANT58 concentration in the tumor-associated bone relative to untargeted NPs, and also provided benefit through the direct antiresorptive therapeutic function of Aln. The dual benefit of the Aln in the BTNPs was supported by the observations that drug-free Aln-containing BTNPs improved bone volume fraction in bone-tumor-bearing mice, while GANT58-BTNPs created better therapeutic outcomes than both unloaded BTNPs and GANT58-loaded untargeted NPs. These findings suggest GANT58-BTNPs have potential to potently inhibit tumor-driven osteoclast activation and resultant bone destruction in patients with bone-associated tumor metastases.

Systemic delivery of a Gli inhibitor via polymeric nanocarriers inhibits tumor-induced bone disease.

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

Settable polymer/ceramic composite bone grafts stabilize weight-bearing tibial plateau slot defects and integrate with host bone in an ovine model.

Bone fractures at weight-bearing sites are challenging to treat due to the difficulty in maintaining articular congruency. An ideal biomaterial for fracture repair near articulating joints sets rapidly after implantation, stabilizes the fracture with minimal rigid implants, stimulates new bone formation, and remodels at a rate that maintains osseous integrity. Consequently, the design of biomaterials that mechanically stabilize fractures while remodeling to form new bone is an unmet challenge in bone tissue engineering. In this study, we investigated remodeling of resorbable bone cements in a stringent model of mechanically loaded tibial plateau defects in sheep. Nanocrystalline hydroxyapatite-poly(ester urethane) (nHA-PEUR) hybrid polymers were augmented with either ceramic granules (85% ß-tricalcium phosphate/15% hydroxyapatite, CG) or a blend of CG and bioactive glass (BG) particles to form a settable bone cement. The initial compressive strength and fatigue properties of the cements were comparable to those of non-resorbable poly(methyl methacrylate) bone cement. In animals that tolerated the initial few weeks of early weight-bearing, CG/nHA-PEUR cements mechanically stabilized the tibial plateau defects and remodeled to form new bone at 16 weeks. In contrast, cements incorporating BG particles resorbed with fibrous tissue filling the defect. Furthermore, CG/nHA-PEUR cements remodeled significantly faster at the full weight-bearing tibial plateau site compared to the mechanically protected femoral condyle site in the same animal. These findings are the first to report a settable bone cement that remodels to form new bone while providing mechanical stability in a stringent large animal model of weight-bearing bone defects near an articulating joint.

3D Printing of Tissue Engineered Constructs for In Vitro Modeling of Disease Progression and Drug Screening.

2D cell culture and preclinical animal models have traditionally been implemented for investigating the underlying cellular mechanisms of human disease progression. However, the increasing significance of 3D vs. 2D cell culture has initiated a new era in cell culture research in which 3D in vitro models are emerging as a bridge between traditional 2D cell culture and in vivo animal models. Additive manufacturing (AM, also known as 3D printing), defined as the layer-by-layer fabrication of parts directed by digital information from a 3D computer-aided design file, offers the advantages of simultaneous rapid prototyping and biofunctionalization as well as the precise placement of cells and extracellular matrix with high resolution. In this review, we highlight recent advances in 3D printing of tissue engineered constructs that recapitulate the physical and cellular properties of the tissue microenvironment for investigating mechanisms of disease progression and for screening drugs.

Fabrication of Trabecular Bone-Templated Tissue-Engineered Constructs by 3D Inkjet Printing.

3D printing enables the creation of scaffolds with precisely controlled morphometric properties for multiple tissue types, including musculoskeletal tissues such as cartilage and bone. Computed tomography (CT) imaging has been combined with 3D printing to fabricate anatomically scaled patient-specific scaffolds for bone regeneration. However, anatomically scaled scaffolds typically lack sufficient resolution to recapitulate the <100 micrometer-scale trabecular architecture essential for investigating the cellular response to the morphometric properties of bone. In this study, it is hypothesized that the architecture of trabecular bone regulates osteoblast differentiation and mineralization. To test this hypothesis, human bone-templated 3D constructs are fabricated via a new micro-CT/3D inkjet printing process. It is shown that this process reproducibly fabricates bone-templated constructs that recapitulate the anatomic site-specific morphometric properties of trabecular bone. A significant correlation is observed between the structure model index (a morphometric parameter related to surface curvature) and the degree of mineralization of human mesenchymal stem cells, with more concave surfaces promoting more extensive osteoblast differentiation and mineralization compared to predominately convex surfaces. These findings highlight the significant effects of trabecular architecture on osteoblast function.