Martin Lab / Tissue Engineering

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Due to life style changes and ageing of our industrialized nations, bone traumatic injuries and osteoporosis induced fragile fracture are an enormous medical and socio-economic challenge. State-of-the-art therapies have failed until now in keeping their promises of reliable bone regenerative solutions. The cmRNAbone project aim to create a novel bone regenerative therapeutic approach based on combination of chemically modified RNAs (cmRNAs)-vectors embedded in a 3D-printed guiding biomaterial ink tailored to patients need. To achieve our goal, sema3a, vegf, pdgf-bb and bmp7 cmRNAs targeting neurogenesis, vasculogenesis and osteogenesis will be synthesized, vectors based on lipids and polysaccharide nanocapsules for the delivery of cmRNAs will be developed. A functional Hyaluronan-Calcium Phosphate biomaterial ink that 1) can be loaded with cmRNAs-vectors and release them, 2) having intrinsic osteoinductivity and presenting laminin-derived peptides for guiding sensory neurons and endothelial cells ingrowth, and 3) being amenable to an extrusion-based 3D-bioprinting process will be formulated in conjunction to a 3D-printer for fabrication of patient specific regenerative solution. In the following step, a large effort will focus on deciphering regenerative mechanisms and optimizing dosage and ratio of cmRNAs, loading of cmRNAs-vectors in the ink, 3D-printing, etc, to demonstrate regenerative capabilities in vitro and in vivo. Selected candidate formulations will be taken to clinically relevant preclinical proof of concepts. Finally, an overreaching effort on preparing a 1st in human trial will be taken, consisting on partners facilities auditing and clinical experts group support, etc, to ensure that GMP-like production for all regenerative tools, and regulatory and commercial strategies are realized.

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.

Verletzungsbedingte und degenerative skeletale Krankheiten, die derzeit meist chirurgisch behandelt werden, könnten auch mit funktionellen skeletalen Ersatzgeweben behandelt werden. Durch die Nutzung von genetischen Modellen der Maus und den Vergleich des Maustranskriptoms von mesenchymalen Vorläuferzellen der Gliedmassen (LMP) mit Maus- und humanen MSCs, wollen wir (i) die gemeinsame moleculare Signatur zwischen LMP und MSCs identifizieren, (ii) die Marker für die Selektion von MSCs mit robustem chondrogenem Potential definieren, und (iii) eine entwicklungsinspirierte Strategie zur Züchtung von Knorpeltemplates entwickeln. Dadurch wird könnte es möglich sein Subpopulationen mit definierten funktionellen Eigenschaften zu identifizieren und stabile Protokolle für die Gewebezüchtung zu etablieren.