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

Research Project
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01.08.2018
 - 31.07.2022

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.

Funding

Engineering of cell-free Extracellular Matrices enriched with osteoinductive and immunomodulatory factors to enhance bone healing

SNF Schweizerischer Nationalfonds, 08.2018-07.2022 (48)
PI : Martin, Ivan.

Members (2)

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Ivan Martin

Principal Investigator
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Andres Garcia-Garcia

Project Member