Martin Lab / Tissue EngineeringHead of Research Unit PD Dr. med. Tarek Ismail, PD Dr. med. Prof. Dr. Ivan Martin, Prof. Dr. PD Dr. med. Arne Mehrkens, PD Dr. med.OverviewMembersPublicationsProjects & CollaborationsProjects & Collaborations OverviewMembersPublicationsProjects & Collaborations Projects & Collaborations 13 foundShow per page10 10 20 50 Development of an in situ strategy for articular cartilage repair through nasal chondrocytes-inspired regenerative molecules Research Project | 1 Project MembersImported from Grants Tool 4722881 Innovative Technologies in Facial Procedures: Objective Measurements and Patient Outcomes Research Project | 5 Project MembersA clinical study evaluating functional and aesthetic outcomes following facial palsy reconstruction Engineering patient-specific bone marrow avatars for personalized medicine in blood cancers Research Project | 1 Project MembersImported from Grants Tool 4721915 Bioengineering native osteochondral architecture: from in vitro models to biological joint resurfacing Research Project | 1 Project MembersImported from Grants Tool 4708801 Unravelling the role of epigenetic modulation on metabolism in human tendinopathies Research Project | 1 Project MembersImported from Grants Tool 4683320 cmRNAbone - 3D Printed-Matrix Assisted Chemically Modified RNAs Bone Regenerative Therapy for Trauma and Osteoporotic Patients Research Project | 3 Project MembersDue 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. Engineering of cell-free extracellular matrices enriched with osteoinductive and immunomodulatory factors to enhance bone healing Research Project | 2 Project MembersInfection, 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. Developmental Engineering of Cartilage from Adult MSCs - Mimicking Differentiation of Limb Mesenchymal Progenitors Research Project | 3 Project MembersVerletzungsbedingte 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. Modulation of pre-vascularization in osteogenic tissue engineered grafts Research Project | 3 Project MembersBackground. Promoting an efficient vascularization of tissue-engineered (TE) osteogenic constructs upon in vivo implantation remains a major challenge towards their clinical application for bone regeneration and repair. Though numerous studies based on pre-vascularization of TE grafts by using vascular cells demonstrated the validity of this approach, they so far failed to define precise parameters, such as the cellular composition (density of endothelial and pericytic cells and of macrophages) and the degree of maturation/ramification of the preformed vascular structures necessary to make such pre-vascular structures fully supportive of vascularization, engraftment and survival of cell inside the implant upon in vivo implantation. This is likely to ultimately regulate tissue formation in vivo. Working hypothesis. The degree of pre-vascularization and the density of macrophages in vitro regulate the vascularization, the engraftment and the bone formation capacity of TE osteogenic grafts in vivo. Specific aims. To address this question, 3 aims are defined: 1) Aim 1 will define if, and how, different in vitro maturation/ramification levels of vascular structures in vitro could affect the density (number of capillaries, branches and total capillary length) of the vascularization in those grafts upon in vivo implantation. 2) Aim 2 will investigate to which extent such different densities of vascularization reached in vivo could in turn affect the engraftment, as judged by cell survival in the graft and performance of the osteogenic grafts in terms of density and volume of bone formation. 3) Finally, aim 3 will examine how macrophages of different phenotypes, included in the grafts or recruited from the host, affect vascularization and bone formation, and how to better harness the power of macrophages to produce enhanced TE bone. Experimental design. The study will use a previously established cell culture and in vivo system using progenitor cells freshly isolated from human adipose tissue, typically referred to as stromal vascular fraction (SVF) cells, demonstrated not to only generate osteogenic grafts but also an intrinsic vasculogenic capacity in vivo. Pre-vascularization density in vitro will be modulated by varying the in vitro culture duration and the effect of this pre-vascularization density on the vascular density, survival of cells inside the implanted grafts and volume of bone formation in vivo. In the same model, the contribution of macrophages of different phenotypes to those processes will be evaluated by depletion of specific populations by using cell sorting or enrichment with peripheral-blood derived macrophages. Expected value of the proposed project. We propose a systematic approach to aim at defining release criteria and quality markers for the use of SVF cells-based grafts as clinical tools, such as i) percentage of specific vascular cells, ii) degrees of vascular cells organization/vascular structure density and iii) percentage and phenotype of macrophages. This is an essential step to initiate preclinical scaled-up models and to pave the way to a pilot clinical trial based on the use of SVF cells-based grafts. Cellular and molecular characterisation of human nasal chondrocyte plasticity, towards their exploration for articular cartilage repair Research Project | 3 Project MembersTrauma and disease of joints frequently involve structural damage of the articular cartilage. These pathologies result in severe pain and disability for millions of people world-wide and represent a major challenge for the orthopaedic community. Cellular therapy and tissue engineering are promising strategies for the repair of such defects. These approaches currently rely on the use of autologous articular chondrocytes (AC), harvested from a small articular cartilage biopsy. As compared to AC, nasal chondrocytes (NC) have been shown to have a higher and more reproducible chondrogenic capacity, thus their clinical utilization would improve the clinical outcome. However, the different embryologic origin of AC and NC (neuroectodermal and mesodermal, respectively), could represent a limit. This study generally addresses the suitability of NC for articular cartilage repair. In particular, modification of the expression of developmentally related HOX genes by NC under controlled conditions will be studied since these genes are supposed to be critical determinants for graft integration into new locations. The working hypothesis of the proposed research is that de-differentiated nasal chondrocytes, by displaying a large degree of plasticity, are compatible with articular joint implantation. The plasticity is defined by the capacity of nasal chondrocytes to (i) exhibit multipotential differentiation capacity -even at clonal levels, (ii) modify their original HOX gene expression status acquiring a pattern closer to that of articular chondrocytes under culture conditions mimicking the natural joint environment and (iii) promote cartilage repair following orthotopic implantation in articular defects created in goats. The proposed research will acquire crucial information about the biology of nasal chondrocytes and their response to specific in vitro conditions mimicking the articular joint environment. Moreover it will provide the necessary pre-clinical data for the potential use of nasal chondrocytes as a cell source for the repair of joint cartilage lesions. Finally, the analysis of the modulation of HOX genes in our cell culture system will represent a first step towards bringing together two traditionally separated disciplines, namely regenerative medicine and developmental biology. 12 12 OverviewMembersPublicationsProjects & Collaborations
Head of Research Unit PD Dr. med. Tarek Ismail, PD Dr. med. Prof. Dr. Ivan Martin, Prof. Dr. PD Dr. med. Arne Mehrkens, PD Dr. med.
Projects & Collaborations 13 foundShow per page10 10 20 50 Development of an in situ strategy for articular cartilage repair through nasal chondrocytes-inspired regenerative molecules Research Project | 1 Project MembersImported from Grants Tool 4722881 Innovative Technologies in Facial Procedures: Objective Measurements and Patient Outcomes Research Project | 5 Project MembersA clinical study evaluating functional and aesthetic outcomes following facial palsy reconstruction Engineering patient-specific bone marrow avatars for personalized medicine in blood cancers Research Project | 1 Project MembersImported from Grants Tool 4721915 Bioengineering native osteochondral architecture: from in vitro models to biological joint resurfacing Research Project | 1 Project MembersImported from Grants Tool 4708801 Unravelling the role of epigenetic modulation on metabolism in human tendinopathies Research Project | 1 Project MembersImported from Grants Tool 4683320 cmRNAbone - 3D Printed-Matrix Assisted Chemically Modified RNAs Bone Regenerative Therapy for Trauma and Osteoporotic Patients Research Project | 3 Project MembersDue 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. Engineering of cell-free extracellular matrices enriched with osteoinductive and immunomodulatory factors to enhance bone healing Research Project | 2 Project MembersInfection, 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. Developmental Engineering of Cartilage from Adult MSCs - Mimicking Differentiation of Limb Mesenchymal Progenitors Research Project | 3 Project MembersVerletzungsbedingte 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. Modulation of pre-vascularization in osteogenic tissue engineered grafts Research Project | 3 Project MembersBackground. Promoting an efficient vascularization of tissue-engineered (TE) osteogenic constructs upon in vivo implantation remains a major challenge towards their clinical application for bone regeneration and repair. Though numerous studies based on pre-vascularization of TE grafts by using vascular cells demonstrated the validity of this approach, they so far failed to define precise parameters, such as the cellular composition (density of endothelial and pericytic cells and of macrophages) and the degree of maturation/ramification of the preformed vascular structures necessary to make such pre-vascular structures fully supportive of vascularization, engraftment and survival of cell inside the implant upon in vivo implantation. This is likely to ultimately regulate tissue formation in vivo. Working hypothesis. The degree of pre-vascularization and the density of macrophages in vitro regulate the vascularization, the engraftment and the bone formation capacity of TE osteogenic grafts in vivo. Specific aims. To address this question, 3 aims are defined: 1) Aim 1 will define if, and how, different in vitro maturation/ramification levels of vascular structures in vitro could affect the density (number of capillaries, branches and total capillary length) of the vascularization in those grafts upon in vivo implantation. 2) Aim 2 will investigate to which extent such different densities of vascularization reached in vivo could in turn affect the engraftment, as judged by cell survival in the graft and performance of the osteogenic grafts in terms of density and volume of bone formation. 3) Finally, aim 3 will examine how macrophages of different phenotypes, included in the grafts or recruited from the host, affect vascularization and bone formation, and how to better harness the power of macrophages to produce enhanced TE bone. Experimental design. The study will use a previously established cell culture and in vivo system using progenitor cells freshly isolated from human adipose tissue, typically referred to as stromal vascular fraction (SVF) cells, demonstrated not to only generate osteogenic grafts but also an intrinsic vasculogenic capacity in vivo. Pre-vascularization density in vitro will be modulated by varying the in vitro culture duration and the effect of this pre-vascularization density on the vascular density, survival of cells inside the implanted grafts and volume of bone formation in vivo. In the same model, the contribution of macrophages of different phenotypes to those processes will be evaluated by depletion of specific populations by using cell sorting or enrichment with peripheral-blood derived macrophages. Expected value of the proposed project. We propose a systematic approach to aim at defining release criteria and quality markers for the use of SVF cells-based grafts as clinical tools, such as i) percentage of specific vascular cells, ii) degrees of vascular cells organization/vascular structure density and iii) percentage and phenotype of macrophages. This is an essential step to initiate preclinical scaled-up models and to pave the way to a pilot clinical trial based on the use of SVF cells-based grafts. Cellular and molecular characterisation of human nasal chondrocyte plasticity, towards their exploration for articular cartilage repair Research Project | 3 Project MembersTrauma and disease of joints frequently involve structural damage of the articular cartilage. These pathologies result in severe pain and disability for millions of people world-wide and represent a major challenge for the orthopaedic community. Cellular therapy and tissue engineering are promising strategies for the repair of such defects. These approaches currently rely on the use of autologous articular chondrocytes (AC), harvested from a small articular cartilage biopsy. As compared to AC, nasal chondrocytes (NC) have been shown to have a higher and more reproducible chondrogenic capacity, thus their clinical utilization would improve the clinical outcome. However, the different embryologic origin of AC and NC (neuroectodermal and mesodermal, respectively), could represent a limit. This study generally addresses the suitability of NC for articular cartilage repair. In particular, modification of the expression of developmentally related HOX genes by NC under controlled conditions will be studied since these genes are supposed to be critical determinants for graft integration into new locations. The working hypothesis of the proposed research is that de-differentiated nasal chondrocytes, by displaying a large degree of plasticity, are compatible with articular joint implantation. The plasticity is defined by the capacity of nasal chondrocytes to (i) exhibit multipotential differentiation capacity -even at clonal levels, (ii) modify their original HOX gene expression status acquiring a pattern closer to that of articular chondrocytes under culture conditions mimicking the natural joint environment and (iii) promote cartilage repair following orthotopic implantation in articular defects created in goats. The proposed research will acquire crucial information about the biology of nasal chondrocytes and their response to specific in vitro conditions mimicking the articular joint environment. Moreover it will provide the necessary pre-clinical data for the potential use of nasal chondrocytes as a cell source for the repair of joint cartilage lesions. Finally, the analysis of the modulation of HOX genes in our cell culture system will represent a first step towards bringing together two traditionally separated disciplines, namely regenerative medicine and developmental biology. 12 12
Development of an in situ strategy for articular cartilage repair through nasal chondrocytes-inspired regenerative molecules Research Project | 1 Project MembersImported from Grants Tool 4722881
Innovative Technologies in Facial Procedures: Objective Measurements and Patient Outcomes Research Project | 5 Project MembersA clinical study evaluating functional and aesthetic outcomes following facial palsy reconstruction
Engineering patient-specific bone marrow avatars for personalized medicine in blood cancers Research Project | 1 Project MembersImported from Grants Tool 4721915
Bioengineering native osteochondral architecture: from in vitro models to biological joint resurfacing Research Project | 1 Project MembersImported from Grants Tool 4708801
Unravelling the role of epigenetic modulation on metabolism in human tendinopathies Research Project | 1 Project MembersImported from Grants Tool 4683320
cmRNAbone - 3D Printed-Matrix Assisted Chemically Modified RNAs Bone Regenerative Therapy for Trauma and Osteoporotic Patients Research Project | 3 Project MembersDue 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.
Engineering of cell-free extracellular matrices enriched with osteoinductive and immunomodulatory factors to enhance bone healing Research Project | 2 Project MembersInfection, 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.
Developmental Engineering of Cartilage from Adult MSCs - Mimicking Differentiation of Limb Mesenchymal Progenitors Research Project | 3 Project MembersVerletzungsbedingte 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.
Modulation of pre-vascularization in osteogenic tissue engineered grafts Research Project | 3 Project MembersBackground. Promoting an efficient vascularization of tissue-engineered (TE) osteogenic constructs upon in vivo implantation remains a major challenge towards their clinical application for bone regeneration and repair. Though numerous studies based on pre-vascularization of TE grafts by using vascular cells demonstrated the validity of this approach, they so far failed to define precise parameters, such as the cellular composition (density of endothelial and pericytic cells and of macrophages) and the degree of maturation/ramification of the preformed vascular structures necessary to make such pre-vascular structures fully supportive of vascularization, engraftment and survival of cell inside the implant upon in vivo implantation. This is likely to ultimately regulate tissue formation in vivo. Working hypothesis. The degree of pre-vascularization and the density of macrophages in vitro regulate the vascularization, the engraftment and the bone formation capacity of TE osteogenic grafts in vivo. Specific aims. To address this question, 3 aims are defined: 1) Aim 1 will define if, and how, different in vitro maturation/ramification levels of vascular structures in vitro could affect the density (number of capillaries, branches and total capillary length) of the vascularization in those grafts upon in vivo implantation. 2) Aim 2 will investigate to which extent such different densities of vascularization reached in vivo could in turn affect the engraftment, as judged by cell survival in the graft and performance of the osteogenic grafts in terms of density and volume of bone formation. 3) Finally, aim 3 will examine how macrophages of different phenotypes, included in the grafts or recruited from the host, affect vascularization and bone formation, and how to better harness the power of macrophages to produce enhanced TE bone. Experimental design. The study will use a previously established cell culture and in vivo system using progenitor cells freshly isolated from human adipose tissue, typically referred to as stromal vascular fraction (SVF) cells, demonstrated not to only generate osteogenic grafts but also an intrinsic vasculogenic capacity in vivo. Pre-vascularization density in vitro will be modulated by varying the in vitro culture duration and the effect of this pre-vascularization density on the vascular density, survival of cells inside the implanted grafts and volume of bone formation in vivo. In the same model, the contribution of macrophages of different phenotypes to those processes will be evaluated by depletion of specific populations by using cell sorting or enrichment with peripheral-blood derived macrophages. Expected value of the proposed project. We propose a systematic approach to aim at defining release criteria and quality markers for the use of SVF cells-based grafts as clinical tools, such as i) percentage of specific vascular cells, ii) degrees of vascular cells organization/vascular structure density and iii) percentage and phenotype of macrophages. This is an essential step to initiate preclinical scaled-up models and to pave the way to a pilot clinical trial based on the use of SVF cells-based grafts.
Cellular and molecular characterisation of human nasal chondrocyte plasticity, towards their exploration for articular cartilage repair Research Project | 3 Project MembersTrauma and disease of joints frequently involve structural damage of the articular cartilage. These pathologies result in severe pain and disability for millions of people world-wide and represent a major challenge for the orthopaedic community. Cellular therapy and tissue engineering are promising strategies for the repair of such defects. These approaches currently rely on the use of autologous articular chondrocytes (AC), harvested from a small articular cartilage biopsy. As compared to AC, nasal chondrocytes (NC) have been shown to have a higher and more reproducible chondrogenic capacity, thus their clinical utilization would improve the clinical outcome. However, the different embryologic origin of AC and NC (neuroectodermal and mesodermal, respectively), could represent a limit. This study generally addresses the suitability of NC for articular cartilage repair. In particular, modification of the expression of developmentally related HOX genes by NC under controlled conditions will be studied since these genes are supposed to be critical determinants for graft integration into new locations. The working hypothesis of the proposed research is that de-differentiated nasal chondrocytes, by displaying a large degree of plasticity, are compatible with articular joint implantation. The plasticity is defined by the capacity of nasal chondrocytes to (i) exhibit multipotential differentiation capacity -even at clonal levels, (ii) modify their original HOX gene expression status acquiring a pattern closer to that of articular chondrocytes under culture conditions mimicking the natural joint environment and (iii) promote cartilage repair following orthotopic implantation in articular defects created in goats. The proposed research will acquire crucial information about the biology of nasal chondrocytes and their response to specific in vitro conditions mimicking the articular joint environment. Moreover it will provide the necessary pre-clinical data for the potential use of nasal chondrocytes as a cell source for the repair of joint cartilage lesions. Finally, the analysis of the modulation of HOX genes in our cell culture system will represent a first step towards bringing together two traditionally separated disciplines, namely regenerative medicine and developmental biology.
