3D printing of heart valves.

3D printing technologies have the potential to revolutionize the manufacture of heart valves through the ability to create bespoke, complex constructs. In light of recent technological advances, we review the progress made towards 3D printing of heart valves, focusing on studies that have utilised these technologies beyond manufacturing patient-specific moulds. We first overview the key requirements of a heart valve to assess functionality. We then present the 3D printing technologies used to engineer heart valves. By referencing International Organisation for Standardisation (ISO) Standard 5840 (Cardiovascular implants - Cardiac valve prostheses), we provide insight into the achieved functionality of these valves. Overall, 3D printing promises to have a significant positive impact on the creation of artificial heart valves and potentially unlock full complex functionality.

Silane-modified hydroxyapatite nanoparticles incorporated into polydioxanone/poly(lactide-co-caprolactone) creates a novel toughened nanocomposite with improved material properties and in vivo inflammatory responses.

The interface tissue between bone and soft tissues, such as tendon and ligament (TL), is highly prone to injury. Although different biomaterials have been developed for TL regeneration, few address the challenges of the TL-bone interface. Here, we aim to develop novel hybrid nanocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL), and hydroxyapatite (HA) nanoparticles suitable for TL-bone interface repair. Nanocomposites, containing 3-10% of both unmodified and chemically modified hydroxyapatite (mHA) with a silane coupling agent. We then explored biocompatibility through in vitro and in vivo studies using a subcutaneous mouse model. Through different characterisation tests, we found that mHA increases tensile properties, creates rougher surfaces, and reduces crystallinity and hydrophilicity. Morphological observations indicate that mHA nanoparticles are attracted by PDO rather than LCL phase, resulting in a higher degradation rate for mHA group. We found that adding the 5% of nanoparticles gives a balance between the properties. In vitro experiments show that osteoblasts' activities are more affected by increasing the nanoparticle content compared with fibroblasts. Animal studies indicate that both HA and mHA nanoparticles (10%) can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. In summary, this work highlights the potential of PDO/LCL/HA nanocomposites as an excellent biomaterial for TL-bone interface tissue engineering applications.

Three-Dimensional Bioprinting in Cardiovascular Disease: Current Status and Future Directions.

Three-dimensional (3D) printing plays an important role in cardiovascular disease through the use of personalised models that replicate the normal anatomy and its pathology with high accuracy and reliability. While 3D printed heart and vascular models have been shown to improve medical education, preoperative planning and simulation of cardiac procedures, as well as to enhance communication with patients, 3D bioprinting represents a potential advancement of 3D printing technology by allowing the printing of cellular or biological components, functional tissues and organs that can be used in a variety of applications in cardiovascular disease. Recent advances in bioprinting technology have shown the ability to support vascularisation of large-scale constructs with enhanced biocompatibility and structural stability, thus creating opportunities to replace damaged tissues or organs. In this review, we provide an overview of the use of 3D bioprinting in cardiovascular disease with a focus on technologies and applications in cardiac tissues, vascular constructs and grafts, heart valves and myocardium. Limitations and future research directions are highlighted.

Novel hybrid biocomposites for tendon grafts: The addition of silk to polydioxanone and poly(lactide-co-caprolactone) enhances material properties, in vitro and in vivo biocompatibility.

Biopolymers play a critical role as scaffolds used in tendon and ligament (TL) regeneration. Although advanced biopolymer materials have been proposed with optimised mechanical properties, biocompatibility, degradation, and processability, it is still challenging to find the right balance between these properties. Here, we aim to develop novel hybrid biocomposites based on poly(p-dioxanone) (PDO), poly(lactide-co-caprolactone) (LCL) and silk to produce high-performance grafts suitable for TL tissue repair. Biocomposites containing 1-15% of silk were studied through a range of characterisation techniques. We then explored biocompatibility through in vitro and in vivo studies using a mouse model. We found that adding up to 5% silk increases the tensile properties, degradation rate and miscibility between PDO and LCL phases without agglomeration of silk inside the composites. Furthermore, addition of silk increases surface roughness and hydrophilicity. In vitro experiments show that the silk improved attachment of tendon-derived stem cells and proliferation over 72 h, while in vivo studies indicate that the silk can reduce the expression of pro-inflammatory cytokines after six weeks of implantation. Finally, we selected a promising biocomposite and created a prototype TL graft based on extruded fibres. We found that the tensile properties of both individual fibres and braided grafts could be suitable for anterior cruciate ligament (ACL) repair applications.

Natural, synthetic and commercially-available biopolymers used to regenerate tendons and ligaments.

Tendon and ligament (TL) injuries affect millions of people annually. Biopolymers play a significant role in TL tissue repair, whether the treatment relies on tissue engineering strategies or using artificial tendon grafts. The biopolymer governs the mechanical properties, biocompatibility, degradation, and fabrication method of the TL scaffold. Many natural, synthetic and hybrid biopolymers have been studied in TL regeneration, often combined with therapeutic agents and minerals to engineer novel scaffold systems. However, most of the advanced biopolymers have not advanced to clinical use yet. Here, we aim to review recent biopolymers and discuss their features for TL tissue engineering. After introducing the properties of the native tissue, we discuss different types of natural, synthetic and hybrid biopolymers used in TL tissue engineering. Then, we review biopolymers used in commercial absorbable and non-absorbable TL grafts. Finally, we explain the challenges and future directions for the development of novel biopolymers in TL regenerative treatment.

Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold.

PURPOSE: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. METHODS: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. RESULTS: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. CONCLUSION: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.

Engineering Heart Valve Interfaces Using Melt Electrowriting: Biomimetic Design Strategies from Multi-Modal Imaging.

Interfaces within biological tissues not only connect different regions but also contribute to the overall functionality of the tissue. This is especially true in the case of the aortic heart valve. Here, melt electrowriting (MEW) is used to engineer complex, user-defined, interfaces for heart valve scaffolds. First, a multi-modal imaging investigation into the interfacial regions of the valve reveals differences in collagen orientation, density, and recruitment in previously unexplored regions including the commissure and inter-leaflet triangle. Overlapping, suturing, and continuous printing methods for interfacing MEW scaffolds are then investigated for their morphological, tensile, and flexural properties, demonstrating the superior performance of continuous interfaces. G-codes for MEW scaffolds with complex interfaces are designed and generated using a novel software and graphical user interface. Finally, a singular MEW scaffold for the interfacial region of the aortic heart valve is presented incorporating continuous interfaces, gradient porosities, variable layer numbers across regions, and tailored fiber orientations inspired by the collagen distribution and orientation from the multi-modal imaging study. The scaffold exhibits similar yield strain, hysteresis, and relaxation behavior to porcine heart valves. This work demonstrates the ability of a bioinspired approach for MEW scaffold design to address the functional complexity of biological tissues.

Fabrication of human myocardium using multidimensional modelling of engineered tissues.

Biofabrication of human tissues has seen a meteoric growth triggered by recent technical advancements such as human induced pluripotent stem cells (hiPSCs) and additive manufacturing. However, generation of cardiac tissue is still hampered by lack of adequate mechanical properties and crucially by the often unpredictable post-fabrication evolution of biological components. In this study we employ melt electrowriting (MEW) and hiPSC-derived cardiac cells to generate fibre-reinforced human cardiac minitissues. These are thoroughly characterized in order to build computational models and simulations able to predict their post-fabrication evolution. Our results show that MEW-based human minitissues display advanced maturation 28 post-generation, with a significant increase in the expression of cardiac genes such as MYL2, GJA5, SCN5A and the MYH7/MYH6 and MYL2/MYL7 ratios. Human iPSC-cardiomyocytes are significantly more aligned within the MEW-based 3D tissues, as compared to conventional 2D controls, and also display greater expression of C×43. These are also correlated with a more mature functionality in the form of faster conduction velocity. We used these data to develop simulations capable of accurately reproducing the experimental performance. In-depth gauging of the structural disposition (cellular alignment) and intercellular connectivity (C×43) allowed us to develop an improved computational model able to predict the relationship between cardiac cell alignment and functional performance. This study lays down the path for advancing in the development ofin silicotools to predict cardiac biofabricated tissue evolution after generation, and maps the route towards more accurate and biomimetic tissue manufacture.

Tissue engineering of corneal stroma via melt electrowriting.

The cornea serves as the main refractive component of the eye with the corneal stroma constituting the thickest component in a stratified layered system of epithelia, stroma, and endothelium. Current treatment options for patients suffering from corneal diseases are limited to transplantation of a human donor cornea (keratoplasty) or to implantation of an artificial cornea (keratoprosthesis). Nevertheless, donor shortage and failure of artificial corneas to integrate with local tissue constitute important problems that have not been yet circumvented. Recent advances in biofabrication have made great progress toward the manufacture of tailored biomaterial templates with the potential of guiding partially or totally the regeneration process of the native cornea. However, the role of the corneal stroma on current tissue engineering strategies is often neglected. Here, we achieved a tissue-engineered corneal stroma substitute culturing primary keratocytes on scaffolds prepared via melt electrowriting (MEW). Scaffolds were designed to contain highly organized micrometric fibers to ensure transparency and encourage primary human keratocytes to self-orchestrate their own extracellular matrix deposition and remodeling. Results demonstrated reliable cell attachment and growth over a period of 5 weeks and confirmed the formation of a dense and highly organized de novo tissue containing collagen I, V, and VI as well as Keratocan, which resembled very closely the native corneal stoma. In summary, MEW brings us closer to the biofabrication of a viable corneal stroma substitute.

Visualization of USPIO-labeled melt-electrowritten scaffolds by non-invasive magnetic resonance imaging.

Melt electrowriting (MEW) is a high-resolution fiber-forming technology for the digital fabrication of complex micro-structured scaffolds for tissue engineering, which has convincingly shown its potential in in vitro and in vivo animal studies. The clinical translation of such constructs to the patient requires the capability to visualize them upon implantation with clinically accepted methods such as magnetic resonance imaging (MRI). To this end, this work presents the modification of polycaprolactone (PCL) scaffolds with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles to render them visualizable by MRI. Composite scaffolds containing up to 0.3 weight % USPIOs were 3D printed by MEW and could be sensitively detected in vitro using T2- and T2*-weighted MRI. At the same time, USPIO incorporation did not affect the usability of PCL for tissue engineering applications as demonstrated by the mechanical and cytocompatibility evaluation. Concentrations up to 0.2% caused small to no decrease in the ultimate tensile strength and Young's modulus. Cytocompatibility tests resulted in excellent cell viability, with proliferating cells adhering to all the scaffolds. This work contributes to the materials library for MEW and opens the possibility of using MRI for longitudinal monitoring of MEW grafts.

Biofabrication and Signaling Strategies for Tendon/Ligament Interfacial Tissue Engineering.

Tendons and ligaments (TL) have poor healing capability, and for serious injuries like tears or ruptures, surgical intervention employing autografts or allografts is usually required. Current tissue replacements are nonideal and can lead to future problems such as high retear rates, poor tissue integration, or heterotopic ossification. Alternatively, tissue engineering strategies are being pursued using biodegradable scaffolds. As tendons connect muscle and bone and ligaments attach bones, the interface of TL with other tissues represent complex structures, and this intricacy must be considered in tissue engineered approaches. In this paper, we review recent biofabrication and signaling strategies for biodegradable polymeric scaffolds for TL interfacial tissue engineering. First, we discuss biodegradable polymeric scaffolds based on the fabrication techniques as well as the target tissue application. Next, we consider the effect of signaling factors, including cell culture, growth factors, and biophysical stimulation. Then, we discuss human clinical studies on TL tissue healing using commercial synthetic scaffolds that have occurred over the past decade. Finally, we highlight the challenges and future directions for biodegradable scaffolds in the field of TL and interface tissue engineering.

Convergence of 3D printed biomimetic wound dressings and adult stem cell therapy.

Biomimetically designed medical-grade polycaprolactone (mPCL) dressings are 3D-printed with pore architecture and anisotropic mechanical characteristics that favor skin wound healing with reduced scarring. Melt electrowritten mPCL dressings are seeded with human gingival tissue multipotent mesenchymal stem/stromal cells and cryopreserved using a clinically approved method. The regenerative potential of fresh or frozen cell-seeded mPCL dressing is compared in a splinted full-thickness excisional wound in a rat model over six weeks. The application of 3D-printed mPCL dressings decreased wound contracture and significantly improved skin regeneration through granulation and re-epithelialization compared to control groups. Combining 3D-printed biomimetic wound dressings and precursor cell delivery enhances physiological wound closure with reduced scar tissue formation.

3D Quantification of Vascular-Like Structures in z Stack Confocal Images.

Optical slice microscopy is commonly used to characterize the morphometric features of 3D cellular cultures, such as in vitro vascularization. However, the quantitative analysis of those structures is often performed on a single 2D maximum intensity projection image, limiting the accuracy of data obtained from 3D cultures. Here, we present a protocol for the quantitative analysis of z stack images, utilizing Fiji, Amira, and WinFiber3D. This protocol facilitates the in-depth examination of vascular-like structures within 3D cell culture models. For complete details on the use and execution of this protocol, please refer to Koch et al. (2020).

Melt Electrowriting of Complex 3D Anatomically Relevant Scaffolds.

The manufacture of fibrous scaffolds with tailored micrometric features and anatomically relevant three-dimensional (3D) geometries for soft tissue engineering applications remains a great challenge. Melt electrowriting (MEW) is an advanced additive manufacturing technique capable of depositing predefined micrometric fibers. However, it has been so far inherently limited to simple planar and tubular scaffold geometries because of the need to avoid polymer jet instabilities. In this work, we surmount the technical boundaries of MEW to enable the manufacture of complex fibrous scaffolds with simultaneous controlled micrometric and patient-specific anatomic features. As an example of complex geometry, aortic root scaffolds featuring the sinuses of Valsalva were realized. By modeling the electric field strength associated with the MEW process for these constructs, we found that the combination of a conductive core mandrel with a non-conductive 3D printed model reproducing the complex geometry minimized the variability of the electric field thus enabling the accurate deposition of fibers. We validated these findings experimentally and leveraged the micrometric resolution of MEW to fabricate unprecedented fibrous aortic root scaffolds with anatomically relevant shapes and biomimetic microstructures and mechanical properties. Furthermore, we demonstrated the fabrication of patient-specific aortic root constructs from the 3D reconstruction of computed tomography clinical data.

Biologically Inspired Scaffolds for Heart Valve Tissue Engineering via Melt Electrowriting.

Heart valves are characterized to be highly flexible yet tough, and exhibit complex deformation characteristics such as nonlinearity, anisotropy, and viscoelasticity, which are, at best, only partially recapitulated in scaffolds for heart valve tissue engineering (HVTE). These biomechanical features are dictated by the structural properties and microarchitecture of the major tissue constituents, in particular collagen fibers. In this study, the unique capabilities of melt electrowriting (MEW) are exploited to create functional scaffolds with highly controlled fibrous microarchitectures mimicking the wavy nature of the collagen fibers and their load-dependent recruitment. Scaffolds with precisely-defined serpentine architectures reproduce the J-shaped strain stiffening, anisotropic and viscoelastic behavior of native heart valve leaflets, as demonstrated by quasistatic and dynamic mechanical characterization. They also support the growth of human vascular smooth muscle cells seeded both directly or encapsulated in fibrin, and promote the deposition of valvular extracellular matrix components. Finally, proof-of-principle MEW trileaflet valves display excellent acute hydrodynamic performance under aortic physiological conditions in a custom-made flow loop. The convergence of MEW and a biomimetic design approach enables a new paradigm for the manufacturing of scaffolds with highly controlled microarchitectures, biocompatibility, and stringent nonlinear and anisotropic mechanical properties required for HVTE.

A Growth Factor-Free Co-Culture System of Osteoblasts and Peripheral Blood Mononuclear Cells for the Evaluation of the Osteogenesis Potential of Melt-Electrowritten Polycaprolactone Scaffolds.

Scaffolds made of biodegradable biomaterials are widely used to guide bone regeneration. Commonly, in vitro assessment of scaffolds' osteogenesis potential has been performed predominantly in monoculture settings. Hence, this study evaluated the potential of an unstimulated, growth factor-free co-culture system comprised of osteoblasts (OB) and peripheral blood mononuclear cells (PBMC) over monoculture of OB as an in vitro platform for screening of bone regeneration potential of scaffolds. Particularly, this study focuses on the osteogenic differentiation and mineralized matrix formation aspects of cells. The study was performed using scaffolds fabricated by means of a melt electrowriting (MEW) technique made of medical-grade polycaprolactone (PCL), with or without a surface coating of calcium phosphate (CaP). Qualitative results, i.e., cell morphology by fluorescence imaging and matrix mineralization by von Kossa staining, indicated the differences in cell behaviours in response to scaffolds' biomaterial. However, no obvious differences were noted between OB and OB+PBMC groups. Hence, quantitative investigation, i.e., alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP) activities, and gene expression were quantitatively evaluated by reverse transcription-polymerase chain reaction (RT-qPCR), were evaluated only of PCL/CaP scaffolds cultured with OB+PBMC, while PCL/CaP scaffolds cultured with OB or PBMC acted as a control. Although this study showed no differences in terms of osteogenic differentiation and ECM mineralization, preliminary qualitative results indicate an obvious difference in the cell/non-mineralized ECM density between scaffolds cultured with OB or OB+PBMC that could be worth further investigation. Collectively, the unstimulated, growth factor-free co-culture (OB+PBMC) system presented in this study could be beneficial for the pre-screening of scaffolds' in vitro bone regeneration potential prior to validation in vivo.

Periosteum-derived mesenchymal progenitor cells in engineered implants promote fracture healing in a critical-size defect rat model.

An attractive alternative to bone autografts is the use of autologous mesenchymal progenitor cells (MSCs) in combination with biomaterials. We compared the therapeutic potential of different sources of mesenchymal stem cells in combination with biomaterials in a bone nonunion model. A critical-size defect was created in Sprague-Dawley rats. Animals were divided into six groups, depending on the treatment to be applied: bone defect was left empty (CTL); treated with live bone allograft (LBA); hrBMP-2 in collagen scaffold (CSBMP2 ); acellular polycaprolactone scaffold (PCL group); PCL scaffold containing periosteum-derived MSCs (PCLPMSCs ) and PCL containing bone marrow-derived MSCs (PCLBMSCs ). To facilitate cell tracking, both MSCs and bone graft were isolated from green fluorescent protein (GFP)-transgenic rats. CTL group did not show any signs of healing during the radiological follow-up (n = 6). In the LBA group, all the animals showed bone bridging (n = 6) whereas in the CSBMP2 group, four out of six animals demonstrated healing. In PCL and PCLPMSCs groups, a reduced number of animals showed radiological healing, whereas no healing was detected in the PCLBMSCs group. Using microcomputed tomography, the bone volume filling the defect was quantified, showing significant new bone formation in the LBA, CSBMP2 , and PCLPMSCs groups when compared with the CTL group. At 10 weeks, GFP positive cells were detected only in the LBA group and restricted to the outer cortical bone in close contact with the periosteum. Tracking of cellular implants demonstrated significant survival of the PMSCs when compared with BMSCs. In conclusion, PMSCs improve bone regeneration being suitable for mimetic autograft design.

Radium 223-Mediated Zonal Cytotoxicity of Prostate Cancer in Bone.

BACKGROUND: Bone-targeting radiotherapy with Radium-223 (Rad-223), a radioisotope emitting genotoxic alpha-radiation with limited tissue penetrance (∼100 µm), prolongs the survival of patients with metastatic prostate cancer (PCa). Confoundingly, the clinical response to Rad-223 is often followed by detrimental relapse and progression, and whether Rad-223 causes tumor-cell directed cytotoxicity in vivo remains unclear. We hypothesized that limited radiation penetrance in situ defines outcome. METHODS: We tested Rad-223 overall response by PC3 and C4-2B human PCa cell lines in mouse bones (n = 5-18 tibiae per group). Rad-223 efficacy at subcellular resolution was determined by intravital microscopy analysis of dual-color fluorescent PC3 cells (n = 3-4 mice per group) in tissue-engineered bone constructs. In vivo data were fed into an in silico model to predict Rad-223 effectiveness in lesions of different sizes (1-27, 306 initial cells; n = 10-100 simulations) and the predictions validated in vivo by treating PCa tumors of varying sizes in bones (n = 10-14 tibiae per group). Statistical tests were performed by two-sided Student t test or by one-way ANOVA followed by Tukey's post-hoc test. RESULTS: Rad-223 (385 kBq/kg) delayed the growth (means [SD]; comparison with control-treated mice) of PC3 (6.7 × 105[4.2 × 105] vs 2.8 × 106 [2.2 × 106], P = .01) and C4-2B tumors in bone (7.7 × 105 [4.0 × 105] vs 3.5 × 106 [1.3 × 106], P < .001). Cancer cell lethality in response to Rad-223 (385 kBq/kg) was profound but zonally confined along the bone interface compared with the more distant tumor core, which remained unperturbed (day 4; 13.1 [2.3%] apoptotic cells, 0-100 µm distance from bone vs 3.6 [0.2%], >300 µm distance; P = .01).In silico simulations predicted greater efficacy of Rad-223 on single-cell lesions (eradication rate: 88.0%) and minimal effects on larger tumors (no eradication, 16.2% growth reduction in tumors of 27 306 cells), as further confirmed in vivo for PC3 and C4-2B tumors. CONCLUSIONS: Micro-tumors showed severe growth delay or eradication in response to Rad-223, whereas macro-tumors persisted and expanded. The relative inefficacy in controlling large tumors points to application of Rad-223 in secondary prevention of early bone-metastatic disease and regimens co-targeting the tumor core.

Printomics: the high-throughput analysis of printing parameters applied to melt electrowriting.

Melt electrowriting (MEW) combines the fundamental principles of electrospinning, a fibre forming technology, and 3D printing. The process, however, is highly complex and the quality of the fabricated structures strongly depends on the interplay of key printing parameter settings including processing temperature, applied voltage, collection speed, and applied pressure. These parameters act in unison, comprising the principal forces on the electrified jet: pushing the viscous polymer out of the nozzle and mechanically and electrostatically dragging it for deposition towards the collector. Although previous studies interpreted the underlying mechanism of electrospinning with polymer melts in a direct writing mode, contemporary devices used in laboratory environments lack the capability to collect large data reproducibly. Yet, a validated large data set is a condition sine qua non to design an in-process control system which allows to computer control the complexity of the MEW process. For this reason, we engineered an advanced automated MEW system with monitoring capabilities to specifically generate large, reproducible data volumes which allows the interpretation of complex process parameters. Additionally, the design of an innovative real-time MEW monitoring system identifies the main effects of the system parameters on the geometry of the fibre flight path. This enables, for the first time, the establishment of a comprehensive correlation between the input parameters and the geometry of a MEW jet. The study verifies the most stable process parameters for the highly reproducible fabrication of a medical-grade poly(ε-caprolactone) fibres and demonstrates how Printomics can be performed for the high throughput analysis of processing parameters for MEW.

Intravital microscopy of osteolytic progression and therapy response of cancer lesions in the bone.

Intravital multiphoton microscopy (iMPM) in mice provides access to cellular and molecular mechanisms of metastatic progression of cancers and the underlying interactions with the tumor stroma. Whereas iMPM of malignant disease has been performed for soft tissues, noninvasive iMPM of solid tumor in the bone is lacking. We combined miniaturized tissue-engineered bone constructs in nude mice with a skin window to noninvasively and repetitively monitor prostate cancer lesions by three-dimensional iMPM. In vivo ossicles developed large central cavities containing mature bone marrow surrounded by a thin cortex and enabled tumor implantation and longitudinal iMPM over weeks. Tumors grew inside the bone cavity and along the cortical bone interface and induced niches of osteoclast activation (focal osteolysis). Interventional bisphosphonate therapy reduced osteoclast kinetics and osteolysis without perturbing tumor growth, indicating dissociation of the tumor-stroma axis. The ossicle window, with its high cavity-to-cortex ratio and long-term functionality, thus allows for the mechanistic dissection of reciprocal epithelial tumor-bone interactions and therapy response.