Additive manufacturing & modelling

In order to build the macro-network, the initial method used by the group of Lorenzo Moroni, Professor of Biofabrication for regenerative medicine at the University of Maastricht and partner in B2B, it’s a hybrid process between bioprinting and additive manufacturing. As he explains “We first create fibers that correspond to the dimensions of the vessels that we want to make. These fibers are made of a material that can be washed out by a water solution; so, once they are embedded in the gels that comprise the beast or the bone compartments, the fibers can be leached away, leaving their trace as a template.” This template is later covered by endothelial cells, thus forming a network of vessels that is expected to behave as a biological unit.

“While the technique in itself is not new, the innovation relies on the level of branching that we wish to obtain in the same construct.” Indeed, the laboratory of Prof. Moroni at MERLN is currently working to build the full network in one manufacturing session, which imply to fine-tune and control of several processing parameters to recreate the required change of dimensions and number of branching.

The design of the branching within the network needs to be carefully planned too. That’s why the Moroni’s lab takes advantage of computational modelling to figure out how to reproduce a physiological vascular tree. “Classical principle of microfluidics not always mimic very well the natural vessels in our body. We have developed an improved model to better understand the physiological level of branching and the architecture of a branched network”.

While computational power might help, it also brings another challenge: translate the physiological features into machine code and extract from a computational model a real prototype.

The B2B semi-synthetic solution

The network in B2B mimics as much as possible the physiological vascular network found in the real tissues, both in the breast and the bone side. One of the strengths and most innovative aspects is the fact that its vessels branch out and change the diameter (see news on mesoscale).

Another feature that brings the device closer to its physiological counterpart is the endothelialisation of the vessels: endothelial cells cover their surfaces making such vessels fully biological. The cells are actually included only after the manufacturing process, once the template is removed (see news on additive manufacturing).

The flow supported by the B2B network will be calibrated as close as possible to the physiological one, with a similar flow rate and shear stress on the covering cell. Otherwise, several problems might arise: turbulence effects and blood clots might cause the formation of micro-thrombi, or a higher flow rate might lead to bulged vessels thus provoking a synthetic-like form of an aneurism. Special care is needed at the level of the bifurcations, as the blood that flows through these regions has a more turbulent flow and the impact on these areas is more vigorous.

Other types of complexity instead fall outside the scope of the B2B project. For the time being in the project, the circulating fluid in the network will be a mix of culture media: the ones typically used in the breast and bone compartments and a classical media that maintains the endothelial cells alive and functional. The resulting media should be able to transport possible metastasis from breast to bone. As a possible future improvement, the cultural media could be substituted with blood, which would increase the physiological biomimicry of the B2B device, but would also bring more challenges related to studying the possible occurring of clotting. For as much as this is an important challenge to tackle in implantable synthetic vascular grafts, it remains of less importance for 3D in vitro models.

The meso scale: bridging the macro & the micro

The macrovascular part of the B2B device is a network aimed at replicating the key features of vascular network found in our body – therefore it includes a smooth transition from relatively large vessel down to micro-vessels.

“To cover the mesoscale that lies in between the macro and the micro scales, that’s where the real challenge is. In B2B, we will try to cover this gap and bridge vessels of few mm with those in the sub-mm scale”, explains Prof. Moroni, head of the Complex Tissue Regeneration department at MERLN and responsible for the development of the macro-network in the B2B device.

Indeed, the current technology allows us to build wonderful scaffolds above the mm scale, and, thanks to the biological angiogenesis, we can well reproduce micro-vessels or capillaries that are below the tens of microns scale. In B2B instead, the network will start from vessels in the order of a few mm of diameter and it will go down to reach a scale of tens of microns. According to Prof. Moroni, “This is a challenge not only from the technological point of view but also from the physiological point of view, as we need to ensure that the bio-fabricated network maintains a correct flow in all the sub-branches of the network.”

He continues: “It’s already quite difficult to have a single vessel which is physiologically functional in the order of few mm.” For example, an unmet clinical need is the creation of an artificial coronary artery (5 mm), for which the solutions developed so far still present some shortcomings as they tend to form blood clots. “We start from this challenge, but in B2B we expand it to embrace multiscale complexity by aiming to manufacture an array of branching vessels of different size.”

Another challenging aspect the B2B technology is the connection between the macro-network and the micro-vessel, the latter created by the group of A. Banfi at University of Basel (read the news here). “It might sound logical but it’s harder than one can imagine, because the two parts have a different nature: one is biological (the capillaries) and the other one comes from a bioengineering approach – therefore, their compatibility and communication is not granted.” The two systems need to merge and create a continuous lumen that runs from the macro to the micro.

The application of the B2B device

The B2B device is a unique environment to study a complex process in a controlled system. In an in vivo study, for example, several factors come into play e.g. the diet of the animal, its immune system, etc., and it’s much harder to define the impact of each one of these variables.

A simplified system like the B2B device helps instead to clear up our view of the process and to focus on key contributing factors. For example, in a controlled situation, it is easier to link a perturbation, as the addition of a drug, to a response without worrying about the influence of other factors. This is essential for the first application that we have envisioned for the B2B device, namely, to test compounds that might inhibit the formation of metastasis.

“In this regard, the B2B device is truly unique because it lets us model the whole process of cancer development, including both the growth of the mass at the primary site and the metastasis formation” as pointed out by Prof. Aceto “Therefore, we can look for anticancer treatments that are not only reducing the mass of the primary tumor but also the number of metastasis found downstream.”

The B2B device might have even wider applications. “It is essentially a chamber that hosts the spontaneous formation of a vascularized tissue – and it doesn’t need to be a cancerogenic tissue.” Indeed, the device can host other tissues and model their pathological or healthy behavior in a 3D setting. The potential is definitely very high.

Following and analysing the metastasis

The main purpose of the B2B device is to follow and dissect the steps of the metastatic process. Therefore, once some cancer cells have left the primary site, we aim to isolate and analyze their features. The main problem during this phase is that within the samples the metastatic cells are rare. “We are talking probably about less than a hundred cells – therefore, we need tools capable to detect and isolate such a small amount.”

The solution is to label the cancer cells before their insertion into the chamber. The labeling can be done with a fluorescent protein (GFP, RFP, etc.) or a luciferase – a protein found in fireflies that is responsible for their ability to emit light. According to Prof. Aceto “Such method allows us to isolate the tumor cells found in samples taken from the circulation of the B2B device. The situation is harder within the bone tissue due to the complexity of the environment. But even then, using microscopic imaging, we can recognize the metastatic cells among the bone ones thanks to their fluorescence or luminescence.”

In the isolated cancer cells, we plan to study the expression levels of some protein markers. In this way we can address specific questions; e.g. in order to understand if the cells are still proliferating while they go through the circulation, we can stain the samples with antibodies that detect proliferation markers. The limitation of this approach is that it relies on a group of pre-selected markers. To obtain a more general characterization of the metastatic cells we plan to perform a whole transcriptome analysis. The sequencing of the RNAs provides a snapshot about which genes are expressed by a cell in a particular moment and their level of expression. By comparing these expression profiles with the ones found in non-metastatic cells (the ones that remain in the primary tumor), we can identify molecular signals that might be responsible for the metastatic behavior. The same information can also be used to infer something about the microenvironment or stresses that have generated such behavior. This knowledge will guide us in the selection of the type of drugs to test as metastatic inhibitors (link to 3rd news – see below).

The breast cancer chamber: as close as possible to reality

In the B2B device, just as in real life, the metastatic process starts within a primary tumor. Indeed, the device hosts a patient-derived breast cancer lesion in a dedicated chamber, which is developed by the Cancer Metastasis group led by Nicola Aceto at the University of Basel.

“We designed the breast cancer chamber so that it is as close to reality as possible”, explains Prof. Aceto. “First, it hosts a 3D growing tumor mass, as opposed to more static systems which usually enable the proliferation only in two dimensions.” In B2B instead, the tumor mass can grow autonomously in three dimensions, mimicking the spontaneous behavior of a tumor. “To increase the resemblance,” continues Nicola, “we opted for patient-derived circulating tumor cells. The classic cancerogenic cell lines have been propagated for so many years that they have adapted over time. Our patient-derived cells are instead grown in the lab for the minimum amount of time and then placed directly into the device, where they continue to grow.”

The B2B breast cancer lesion has also a network of self-assembled capillaries that connect the tissue to a network of larger artificial vessels (more here). To develop these natural capillaries, “we will include within the tumor mass some endothelial cells –cells that naturally form blood vessels – and as the tumor grows, they will grow too and form a microcapillary network.” This network is then put in contact with the macro network, developed by the University of Maastricht. The smooth transition from small natural vessels to big artificial ones is one of the most critical and innovative features of the B2B device (more here).

Finally, thanks to the B2B concept the extravasation of cells is spontaneous and not forced by the system. “We expect that some cancer cells will recognize the presence of blood vessels and they will spontaneously migrate there and join the systemic circulation” says Nicola “While we are fairly positive that this phenomenon will happen, its frequency might depend on several factors e.g. how invasive the primary cells are, how many capillaries there are in the system, what is the micro-environment, etc.” These parameters might be used later on to fine-tune the system and modulate the metastasis frequency.

A CRITICAL KISS: FROM MACRO TO MICRO

While the fabrication of a self-assembled network of capillaries and an engineered macro-network is not trivial but possible, the connection between the two is a real challenge. “The macro-to-micro connection proposed by B2B is truly innovative and, to the best of our knowledge, it’s the first time that it would be implemented” explains Andrea Banfi, leader of the related work package in B2B. The distinguishing factor of B2B, and the one that brings most of the complexity, is the size of the involved tissues, which require an extensive network of capillaries to penetrate the whole mass (range of cm3). In other techniques, such as the so-called “organ-on-a-chip” only a few thousand cells of each kind communicate with each other  – so, the connecting system is greatly simplified.

“An extended network of capillaries is harder to reproduce because its development and final arrangement is linked to conditions evolving in the different micro-environments of the organoid, which cannot be precisely anticipated”, says Banfi, “To reproduce the real organ-level-complexity we should let this randomness take place.”

That’s why in B2B the functional connection between the micro- and macro-networks is built by spontaneous processes that rely on the normal biological behavior of endothelial cells and fluid flow. The endothelial cells that cover the engineered vase and the ones that form the capillaries should first recognize each other, then stick together and finally create a junction with a lumen. For this to happen, “we include in the micro-environment the right biological signals, e.g. growth factors and morphogens, to trigger the cascade of events leading to vessel assembly. Similarly, once connections are established, the fluid flow should naturally regulate and remodel the shape and size of the vascular network.” Overall, this spontaneous connection should not take more than a few days.

After the 1st part of the project, everything is ready to test this critical kiss: the set-up for the micro- and macro-networks have been identified and it’s time to place them together and let the system evolve. The first tests will provide useful feedback to fine-tune the two networks and ensure their compatibility.

TO EACH TISSUE, ITS MICRO-NETWORK

Example of micro-network (capillaries) with endothelial cells in green. The cell nuclei (5 microns on average) are visible in blue or pink (if proliferating). Courtesy of Andrea Banfi.

In B2B, both the breast tumoroid and the ossicle have their self-assembled networks of capillaries.

In the first case, the tumoroid and its vascular network are generated in parallel. “We include cancer cells together with endothelial cells in a matrix of fibrin that contains growth factors for both tissues.” explains Andrea Banfi. Thanks to the biological signals, the system evolves by creating two ordered networks of cells of the same type: endothelial with endothelial and cancer with cancer. However, the two systems are in close contact, meaning that the tumoroid is fully vascularized.

Thanks to the large dimensions of the tumoroid, B2B reproduces well the stochastic growth of the cancer tissue. In some cases, it might exceed the flow capacity of the system, thus leading to the formation of necrotic and hypoxic areas. It is the presence of these areas that increases the metastatic predisposition of cells, as demonstrated by B2B partner Nicola Aceto, as if the lack of resources would trigger the need to migrate to new, richer areas. Only thanks to the peculiarities of the B2B system, based on spontaneous processes, it is possible to recreate such heterogeneity so vital for the selection of cells capable to metastasize (read more here).

Different is the approach for the ossicle, which is generated in vivo – by placing chondrogenic cells subcutaneously in a mouse. The resulting ossicle is then vascularized directly by the mouse system. “When we remove the ossicle, the major blood vessels are cut and we need to re-establish a connection, this time with the macro system“.  This is quite hard as the vessels are placed randomly around the tissue, therefore it would be impossible to engineer something ad hoc.

The solution proposed by the team of Andrea Banfi is to generate a pervasive micro-network (of human origin) that extends over the whole surface around the tissue, touching and connecting, on one side, with the ossicle capillaries and, on the other side, with the macro network. When the connection happens, then the flow, and the physical laws governing it, remodel the dimensions of the used vessels and dismiss the unused ones.

MACRO & MICRO in B2B

3D rendering of a micro-network: endothelial capillaries in red, supporting cells in green. Courtesy of Andrea Banfi.

As stated in the name of the project (from Breast to Bone), B2B relies on the connection between two tissues. Indeed, the two chambers, one with the breast tumoroid and the other with the ossicle, are connected with a system that resembles the physiological blood circulation, a set of vessels that brings the blood with oxygen and nutrients to the organs. As in the cardiovascular system, the B2B connecting network is made of two parts: the micro-network for the exchange of material from the tissue to the circulatory system and the macro network for its fast transportation from one side to another.

“As part of the micro-network it was crucial to include the capillaries” – explains Andrea Banfi, head of the Cell and Gene Therapy group at Basel University Hospital and the B2B partner responsible for the micro-network – “Two key events happen only there: the intra- and extravasation of the metastatic cells, the process by which cancer cells move, respectively, from the main tumor to the blood circulation and from the circulation to the target tissue”.

The two processes happen only in the smallest vessels (10-20 micrometers of caliber) because here the blood flow is slow enough to allow metastatic cells to roll and adhere to the blood vessels’ surface, the endothelium, and leak out. Instead, in larger vessels, the higher velocity of the flow hinders cell movement through the vessel wall. Also, structurally the capillaries encourage the exchange of cells and substances: they are made of a thin layer of mainly endothelial cells – so, to exit, a metastatic cell just needs to squeeze through the gaps between them. However, the small dimensions of the capillaries make them non-engineerable: therefore, they need to self-assemble under the guidance of provided biological signals and molecules (read more here).

The branching of an artery-vein pair. Courtesy of Andrea Banfi.

The organoid chambers are connected by large macro-fluidic tubing, made of silicone, whose function is equivalent to that of large arteries and veins in the body, whose flux quickly transports substances from one side to the other. To bring flow from this tubing system to the self-assembled micro-vessels, an actual vascular network of decreasing size and increasing branching is required. This is the macro-vascular network, which is built by a set of engineered vessels bio-printed into and around the micro-vascularized organoids, whose diameters gradually range from large to small. This system follows the same laws that regulate the relationship between flow and dimension in the cardiovascular tree. But, unlike the self-assembled capillaries, at the moment the bio-printed vessels lack part of the structural features found in the physiological counterpart, like the smooth muscle surrounding arteries and responsible for their elasticity and contraction. This doesn’t impair the scope of the B2B device, as the global flow can be regulated by an external pump.

The integration between the macro- and micro-networks is one of the most innovative points in B2B. The full vascular network is realized in collaboration by two groups: the micro-network by Dr. Andrea Banfi’s lab at the Basel University Hospital and the macro-network by Lorenzo Moroni’s group at MERLN. Their joint efforts will result in an innovative vascular system with a smooth transition from the macro- to the micro-scale (read more here).

Therapeutic Angiogenesis

The B2B partner Andrea Banfi directs the Cell and Gene Therapy group at Basel University Hospital, in the Departments of Biomedicine and of Surgery. His research focus is the understanding of the basic principles governing the growth of blood vessels and translating this knowledge into the development of novel therapies. We asked him to introduce us to the concept of therapeutic angiogenesis.  

New blood vessels forming from pre-existing vessels.
www.scientificanimations.com – http://www.scientificanimations.com/wiki-images/

“Therapeutic angiogenesis is the generation of blood vessels for therapeutic applications. Today, besides the application in tissue-engineering in vitro, like the one in B2B, there is also an increasing interest in the vascularization of ischemic tissues, in which the blood supply is reduced and needs to be restored for the normal organ function.

There are no pharmacological cures for this disease today. Only surgical interventions can substitute blocked arteries (e.g. by-pass surgery), but results are unsatisfactory, both because not every patient can be operated, and because the opened vessels re-close with time. By understanding how angiogenesis is regulated in nature, we might exploit similar signals to trigger new vascular growth directly in the tissue to generate a sort of long-lasting “biological by-pass”.

Common signals are molecules like the growth factor VEGF, but the body tightly regulates their production and avoids exceeding potentially harmful thresholds. To induce therapeutic blood vessels formation, it is necessary to exceed, under limited circumstances, the dose-limit that the body imposes. In our lab, we are investigating the optimal mix of stimuli, doses and duration of treatment to trigger efficient and long-lasting blood vessels formation.

During the very first tests, back in the early 2000s, therapies with VEGF failed to show efficacy in patients at safe doses. Subsequent retrospective analyses identified several issues underlying the discrepancy between the obvious biological function of VEGF and its difficulty as a drug. An important aspect relates to the fact that VEGF binds tightly to extracellular matrix and remains localized in the micro-environment around each producing cell. Therefore, it is important to ensure a homogeneous distribution of production levels in the tissue, otherwise a few hot spots – in which the local dose is toxic – will compromise safety, while the areas that don’t reach an effective dose compromise efficacy.

In our lab, we are currently working to overcome this issue. Homogeneous distributions of VEGF are rather hard to get, so we are exploring ways to stop the onset of the toxic behavior only in the hot spots. By administrating specific drugs during the critical first weeks after the therapy, we can block the aberrant angiogenesis while keeping the desired one. It’s a long way before reaching the clinical application, but I’m confident that we will finally find a way to apply angiogenesis in the fight against ischemia. “

Thank you