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[FG] Technologies for Tissue Engineering

Projects & Collaborations

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Direct 3D Bioprinting strategies to study articular cartilage development and regenerative therapy for osteoarthritis

Research Project  | 2 Project Members

By the year 2030, approximately 25% of the adult population is projected to suffer from clinically diagnosed osteoarthritis (OA). At present OA cannot be treated, but only the associated pain is managed by administration of anti-inflammatory drugs. Implantation of tissue engineered cartilage in the affected lesions has been a hotly pursued research area worldwide for last three decades. However, this line of research has not succeeded so far. The most common problem encountered is that the engineered cartilage generated using mesenchymal progenitor cells (MSCs) does not share the molecular properties of native permanent cartilage but, instead, become vascularized, undergoes hypertrophic differentiation and eventually is replaced by bone once implanted in vivo. These latter features characterize both the transient cartilage (i.e.; cartilage formed at the epiphyseal growth plates of long bones) and the articular cartilage in OA joint. The overall objective of this proposal is to engineer a cartilage tissue which will be molecularly indistinguishable from healthy articular cartilage and which remains phenotypically stable also when implanted in a OA environment. We propose a comprehensive plan to achieve this, at the core of which is to engineer developmental processes in order to control fate specification by MSC and developing functional cartilage tissue construct by 3D bioprinting.Working hypothesis. Most of the tissue engineering studies did not take into account: (i) the natural process of permanent cartilage differentiation; (ii) the molecular niche in native permanent cartilage and the changes it undergoes during OA; (iii) the nature of the engineered cartilage; (iii) the effect that the niche, particularly the altered one in OA, has on tissue engineered cartilage. In the current proposal, we will develop a comprehensive strategy for designing a disease modifying therapy of OA, combining our strengths in 3D bioprinting, biomedical engineering, microfluidics and joint cartilage developmental biology. We hypothesize that phenotypical stable cartilage can be engineered by culturing MSC with signalling proteins (i.e.: Wnt ligands) and morphogenetic inhibitors (BMP inhibitors) recently shown (mainly by the applicants) to be key in regulating differentiation of MSC into stable or transient cartilage. Specific aims & experimental design. The first aim is to generate 3D bioprinted permanent cartilage in vitro. For this purpose, we will initially perform a screening of different doses and temporal stages of supplementation of Wnt ligands and BMP inhibitor within a microfluidic platform allowing generating and perfusing 3D MSCs microaggregates. This will allow identify the most promising conditions enabling the differentiation of MSCs into stable cartilage, to be scaled up and applied into the 3D bioprinted constructs. We will then explore use of silk-gelatin bioink tethered pathway-regulatory molecules on differentiation of the encapsulated mesenchymal cells (Aim 1). Once this is achieved, we will work towards two distinct but complementary approaches: (i) We will develop an in vitro model for OA. For this we will activate BMP signalling in the 3D bioprinted permanent cartilage to investigate if it recapitulates changes associated with OA. This will allow us to create a platform for rapid screening of drugs or other bioactive molecules which may be therapeutically relevant (Aim2). We will then develop bioink which will be either sensitive to matrix metalloproteases or mechanical stimulation to release in situ BMP inhibitors to protect the 3D bioprinted permanent cartilage once implanted in an OA joint (induced in mice), environment which would instead promote transient cartilage differentiation (Aim 3).Expected value of the proposed project. Even after 30 years of efforts, fabrication of load bearing and clinically relevant, biochemically equivalent cartilage tissue constructs still remains elusive. We are aware that this is a novel and highly ambitious objective, but collectively we have the necessary expertise and insight to attempt this, which if achieved will surely be a major boost for developing a disease modifying therapy for OA.

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Engineering of cell-free extracellular matrices enriched with osteoinductive and immunomodulatory factors to enhance bone healing

Research Project  | 2 Project Members

Infection, trauma or tumors can generate critical bone defects with compromised regeneration capacity, still not resolvable through clinical gold standard solutions. Extracellular matrices (ECMs) have been proposed for the physiological presentation of various cytokines at sites of tissue repair to activate and regulate endogenous cells. ECMs do not need to be derived from native tissues, but can be tissue-engineered and then devitalized. This provides the potential advantages of standardization (e.g., thanks to the use of cell lines) and of customization (e.g., by transducing cells to overexpress defined factors). ECMs could be engineered to deliver not only osteoinductive factors, but also immunomodulatory signals, which are increasingly being recognized as strong regulators in bone regeneration. Working hypothesis. We hypothesize that engineered and devitalized ECMs, specifically enriched in osteoinductive factors (BMP-2), as well as in cytokines polarizing immune response towards a pro-regenerative phenotype (IL4) and resolving acute inflammation (IL1Ra), can enhance bone regeneration. Specific aims. In order to generate the modular bricks for the engineering of ECMs, cell lines expressing different instructive cues (BMP-2, IL4 and IL1Ra) will be derived from an existing death-inducible human mesenchymal cell line (MSOD) (Aim1). ECMs enriched with different combinations and doses of such cues will be engineered, characterized and tested in a standard ectopic implantation model (Aim2). The capacity of these ECMs in modulating human immune cell recruitment and polarization will be investigated in vitro (Aim3). The effect of signals presented by ECMs on bone repair and the possible role of macrophages and T cells will be investigated in a rat critical sized calvarial defect (Aim4).Experimental design. Three cell lines will be generated by lentivirally transducing the MSOD line to express BMP2, IL4 and IL1Ra under the control of inducible promoters, and characterized in vitro for secretion of factors, proliferation, differentiation capacity and death-induction responsiveness (Aim1). Generated cell lines will be cultured on collagen scaffolds within a perfusion bioreactor to engineer ECMs, enriched in the different instructive signals. Obtained ECMs will be assessed for protein content and release, compatibility with inducible apoptosis and osteoinductive potential (Aim2). The immunomodulatory effect of engineered ECMs will be evaluated in an in vitro human setup based on i) chemokine release profiles, ii) recruitment and polarization of macrophages and iii) macrophage-mediated polarization of T cells (Aim3). The capacity of engineered ECMs to enhance bone repair will be studied in critical sized calvarial bone defects in immunocompetent rats, with or without depletion of T cells (Aim4). Expected value of the proposed project. Our studies will lead to the establishment of a strategy to engineer cell-free customizable materials presenting specific signals crucial for tissue regeneration. Moreover, our research will allow gaining new insights on the possibility to enhance bone healing by modulating inflammation and immunity. Such knowledge will be instrumental to improve the design of biomaterials/drugs in the broader field of regenerative immunology.

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The underestimated role of the human omentum in metastatic spread

Research Project  | 4 Project Members

High-grade serous ovarian (SOC), peritoneal (SPC) and tubal cancers (STC) are diagnosed in 75% of cases at advanced stage disease in women, when 5 year survival is only 20%. All three cancers have the same histological appearance and are diagnosed and treated the same way. However, knowledge is increasing that whilst all three arise via p53 mutations, they origin from different sites and have different genetic drivers. Whilst STC/SOC is defined by having the biggest tumor volume in the tubes/ovaries and only metastatic disease in the omentum, SPC presents with massive omental tumor-load with no invasion into the ovaries or tubes. Until now, there are no published data to support a different origin for SPC because it seems to be specifically located in the omentum. However, unpublished work from us has shown a revolutionary result: on genetic, proteomic and glycomic level, STC/SOC are distinct to SPC and should therefore be diagnosed and especially treated individually. Correct classification of these cancers, identifying the place of origin and their distinct development would change the paradigm that they are all the same disease which is relevant for the treatment regime and possible survival of women.Since SPC develops specifically in the omentum, we aim to (a) reveal the human omental structure in situ and (b) to design, based on the in-situ information, a relevant 3D human model mimicking the development of SPC.Like the omentum, the fallopian tubes and ovaries are covered by mesothelial cells with submesothelial vascular and lymphatic networks where resident macrophages mature; a robust 3D in vivo-like model, which allows the assessment of cancer cell mesothelial clearance and invasion as well as stem cell population studies has not been designed before and is highly innovative. We will use native tissue and for the first-time tissue-engineered omentum including 3D cell printing technology. Molecular analyses will be performed by deconstructing and reconstructing both omental models, systematically exposing the structures to normal tubal epithelial and serous tubal cancer cells. Hereby, we aim to understand the mechanism of invasion and thus develop new therapeutic targets. Together, these results have the potential to invalidate medical textbooks and shift the paradigm of diagnosis and treatment of high grade serous adenocarcinomas of apparently tubal, ovarian and peritoneal origin.