Projects & Collaborations 5 foundShow per page10 10 20 50 Bone prefabrication for osteonecrosis Research Project | 2 Project MembersBackground Avascular necrosis (AVN), also known as osteonecrosis, leads to sclerosis and collapse of bone. The surgical standard uses vascularized, autologous bone grafts but is highly limited by donor site morbidity and availability. Alternative approaches are thus highly required to meet this strong clinical need. Regenerative approaches based on cell therapy with bone marrow-derived cells are not easily standardized and have so far shown variable clinical outcome. We have recently described the generation of an axially-vascularized bone graft substitute, based on freshly isolated, human adipose-derived cells, compatible with an intraoperative approach for bone repair, and demonstrated its potential to revitalize dead bone in a rat model of AVN. Beyond the proof-of-concept, the current limitation of this approach is the yet too limited amount of bone formation inside the axially-vascularized grafts. Working hypothesis The development and use of osteo-inductive, engineered biomaterials, based on an endochondral ossification (ECO) paradigm, combined to an axially-vascularized graft, can generate strong and reproducible bone formation in preclinical animal models of AVN. Specific aims In this project, 3 aims are defined: 1) Aim 1 will compare different approaches to engineer hypertrophic cartilage tissues (HCT) and evaluate their respective ectopic bone formation capacity through an ECO process. The role of integrin receptors and extracellular matrix proteins in this process will be elucidated. 2) Aim 2 will investigate how these HCT could be devitalized while maintaining their matrix properties, in order to generate devitalized osteo-inductive matrices. The performance of such matrices in bone formation and in bone repair will be assessed. 3) Finally, aim 3 will investigate the implementation of such devitalized HCT inside the axiallyvascularized grafts we recently developed and will assess if HCT can induce a more massive, reproducible and homogenous bone formation inside the grafts and in the necrotic bone around it. Experimental design HCT will be generated either by seeding human bone marrow- and adipose-derived mesenchymal stromal cells (MSC) onto collagen sponges, or by fractionating native, human adipose tissue followed by 3 weeks of culture on agarose-plated petri dishes. All constructs types will then be differentiated into HCT, analyzed or implanted ectopically to assess osteogenicity. The role of integrins will be studied by overexpression/silencing with lentiviral transduction/CRISPR-Cas9-mediated knockout. The performance of HCT, devitalized by various techniques (e.g. freeze drying or hydrostatic pressure) will be analyzed in ectopic and orthotopic (calvaria) in vivo models in rodents. Axially-vascularized grafts will be generated as previously described, containing either ceramic granules (current composition) or devitalized, osteo-inductive HCT, and will be tested in the rat model of AVN for revitalization, vascularization and bone formation potential. Expected value of the proposed project The present project is essential to introduce a routine clinical use of axially vascularized osteogenic grafts as prefabricated bone. The knowledge gained in this study will be useful not only to define formulations for AVN applications but also for other surgical bone regeneration strategies. In addition, more fundamental and molecular knowledge will be acquired on ECO by human bone marrow- and adipose- derived MSC, allowing for a better control of MSC differentiation into HCT and thereby a more efficient and reproducible bone formation in vivo. Finally, this project will directly contribute to the definition of a second-generation of osteogenic grafts to be tested clinically, in the form of a clinical trial. NANOTRANSMED: Innovationen in der Nanomedizin - von der Diagnose zur Implantologie Research Project | 4 Project MembersDurch den Rückgriff auf Nanoobjekte soll das Projekt innovative und effektive Lösungen zur Behandlung von Patienten schaffen: - Frühzeitigen Diagnose: Hauptziel ist die Verbesserung der Effizienz von Targeting-Bildgebungssonden, um eine Vielzahl an Krankheiten (Krebs, neurodegenerative Erkrankungen, Entzündungen) frühzeitig zu diagnostizieren. - Individualisierte Behandlung: Entwicklung theranostischer Nanoobjekte, die in der Lage sind, Diagnose und Behandlung auf wirksame Art und Weise zu kombinieren. - Vermeidung von Infektionen: Entwicklung von robusten Antihaftoberflächen, um eine mikrobielle Besiedlung (die in 5% aller Krankenhausbehandlungen auftritt) effektiv zu vermeiden. 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. Generation of a mesenchymal niche - Importance of CD34 Research Project | 2 Project MembersDue to its abundance and easy access, subcutaneous adipose tissue has emerged as a potential source of adult stromal/stem cells for tissue engineering and regenerative medicine applications. The stromal vascular fraction (SVF) of adipose tissue indeed includes progenitor cells, referred to as human adipose stromal/stem cells (hASC) with extensive plasticity and the capacity to differentiate into various musculoskeletal lineages. SVF cells express a membrane protein, CD34, in vivo but CD34 expression by hASC spontaneously decreases over time in culture. Culture-expanded hASC are therefore described as CD34 negative cells. CD34 expression in SVF cells has recently been associated with increased biological potential. Preliminary results from our group show that long-term cultures of SVF cells in Petri dishes without passaging into new dishes at cell confluence, in order to favor cell-extracellular matrix and cell-cell interactions, stably maintained and expanded CD34 expression in roughly 20 % of hASC for up to 8 weeks of culture, and that those hASC positive for CD34 expressed higher levels of markers known to be expressed by stem cells. These data suggest the generation of a highly specific hASC culture system in vitro. The ultimate goal of the present project aims at defining the role of CD34 in the biology, function and in vivo potential of hASC. We therefore hypothesize that the expression of CD34 characterizes a subset of hASC with increased self renewal and differentiation capacities. We plan to study the following different aspects: 1. To study the functional role of CD34 in hASC biology. 2. To investigate the exploitability of the hASC obtained without passaging, by testing their in vivo performance in terms of tissue formation, with an emphasis on bone tissue. 3. To analyze the mechanisms involved in the maintenance of CD34-positive cells, i.e. the possible contribution by soluble factors, extracellular matrix and cell-cell interactions. The study proposed here, based on the promising preliminary results already provided by the new protocol to expand hASC, will result in a more in-depth knowledge about the biology and possible self-renewal capacity of hASC, the maintenance of their in vitro environment, and about some molecular players, of which CD34, which could be pivotal in the control of hASC phenotype, differentiation potential and in vivo capability. Thereby, this fundamental study will greatly impact on the applicability of ASC in regenerative medicine in general, and in bone tissue engineering in particular. Vascularization of tissue engineered grafts Research Project | 2 Project MembersRationale: Vascularization of engineered tissues of clinically relevant sizes is central for cell survival and successful engraftment. In the field of bone regeneration, several studies have established that bone marrow stromal cells, expanded in culture and loaded into porous ceramic scaffolds, are able to form bone tissue, both ectopically and orthotopically. Despite the report of few clinical cases, however, no convincing successes have been achieved in humans, most likely because of lack of sufficient vascular supply, resulting in immediate cell death after implantation. Recent reports in the context of other tissues suggested that addition of vascular cells inside the graft, such as endothelial cells, could improve vascularization and engraftment. Towards this prospective, we have shown that the generation of osteogenic-vasculogenic constructs starting from a single human cell source containing both osteogenic and endothelial cells, namely adipose tissue, is possible. Overall goal: To modulate the relative fractions and level of maturation/organization of human adipose tissue-derived endothelial cell progenitors (EP) and osteoprogenitor cells in a 3D co-culture system and to use the model to investigate the role of a network of EP on the engraftment and bone formation of the resulting engineered construct. Hypothesis: The presence of EP in tissue engineered bone grafts generated by adipose-derived nucleated cells improves and accelerates vascularization of the construct after in vivo implantation, thereby increasing the amount and uniformity of bone tissue formation. Specific aims: 1. Modulate the level of maturation/organization of adipose-derived endothelial cells and osteoprogenitors inside a 3D co-culture system. 2. Investigate the role of an EP network on the vascularization and bone formation of the resulting engineered construct. 3. Investigate the effect of EP in critically sized engineered constructs on cell survival and bone formation in the central core of the grafts. 4. Develop a serum-free medium to support a reproducible and clinically compliant engineering of the defined construct properties. Relevance of the expected results: This project will provide fundamental knowledge on the yet unresolved process by which EP can organize vascular networks and connect to the vasculature of the host in vivo. In the more general field of tissue engineering, it will provide a way to efficiently overcome one of the most critical limitations in current tissue engineering applications, namely vascularization and engraftment of implanted constructs. Finally, this project is very likely to define the starting point for a pilot clinical study using vasculogenic, autologous bone grafts in the treatment of critical size bone defects such as spine fusion or talus reconstruction. A better engraftment, by accelerating the bone healing process, will obviously have a major health and economical significance. 1 1
Bone prefabrication for osteonecrosis Research Project | 2 Project MembersBackground Avascular necrosis (AVN), also known as osteonecrosis, leads to sclerosis and collapse of bone. The surgical standard uses vascularized, autologous bone grafts but is highly limited by donor site morbidity and availability. Alternative approaches are thus highly required to meet this strong clinical need. Regenerative approaches based on cell therapy with bone marrow-derived cells are not easily standardized and have so far shown variable clinical outcome. We have recently described the generation of an axially-vascularized bone graft substitute, based on freshly isolated, human adipose-derived cells, compatible with an intraoperative approach for bone repair, and demonstrated its potential to revitalize dead bone in a rat model of AVN. Beyond the proof-of-concept, the current limitation of this approach is the yet too limited amount of bone formation inside the axially-vascularized grafts. Working hypothesis The development and use of osteo-inductive, engineered biomaterials, based on an endochondral ossification (ECO) paradigm, combined to an axially-vascularized graft, can generate strong and reproducible bone formation in preclinical animal models of AVN. Specific aims In this project, 3 aims are defined: 1) Aim 1 will compare different approaches to engineer hypertrophic cartilage tissues (HCT) and evaluate their respective ectopic bone formation capacity through an ECO process. The role of integrin receptors and extracellular matrix proteins in this process will be elucidated. 2) Aim 2 will investigate how these HCT could be devitalized while maintaining their matrix properties, in order to generate devitalized osteo-inductive matrices. The performance of such matrices in bone formation and in bone repair will be assessed. 3) Finally, aim 3 will investigate the implementation of such devitalized HCT inside the axiallyvascularized grafts we recently developed and will assess if HCT can induce a more massive, reproducible and homogenous bone formation inside the grafts and in the necrotic bone around it. Experimental design HCT will be generated either by seeding human bone marrow- and adipose-derived mesenchymal stromal cells (MSC) onto collagen sponges, or by fractionating native, human adipose tissue followed by 3 weeks of culture on agarose-plated petri dishes. All constructs types will then be differentiated into HCT, analyzed or implanted ectopically to assess osteogenicity. The role of integrins will be studied by overexpression/silencing with lentiviral transduction/CRISPR-Cas9-mediated knockout. The performance of HCT, devitalized by various techniques (e.g. freeze drying or hydrostatic pressure) will be analyzed in ectopic and orthotopic (calvaria) in vivo models in rodents. Axially-vascularized grafts will be generated as previously described, containing either ceramic granules (current composition) or devitalized, osteo-inductive HCT, and will be tested in the rat model of AVN for revitalization, vascularization and bone formation potential. Expected value of the proposed project The present project is essential to introduce a routine clinical use of axially vascularized osteogenic grafts as prefabricated bone. The knowledge gained in this study will be useful not only to define formulations for AVN applications but also for other surgical bone regeneration strategies. In addition, more fundamental and molecular knowledge will be acquired on ECO by human bone marrow- and adipose- derived MSC, allowing for a better control of MSC differentiation into HCT and thereby a more efficient and reproducible bone formation in vivo. Finally, this project will directly contribute to the definition of a second-generation of osteogenic grafts to be tested clinically, in the form of a clinical trial.
NANOTRANSMED: Innovationen in der Nanomedizin - von der Diagnose zur Implantologie Research Project | 4 Project MembersDurch den Rückgriff auf Nanoobjekte soll das Projekt innovative und effektive Lösungen zur Behandlung von Patienten schaffen: - Frühzeitigen Diagnose: Hauptziel ist die Verbesserung der Effizienz von Targeting-Bildgebungssonden, um eine Vielzahl an Krankheiten (Krebs, neurodegenerative Erkrankungen, Entzündungen) frühzeitig zu diagnostizieren. - Individualisierte Behandlung: Entwicklung theranostischer Nanoobjekte, die in der Lage sind, Diagnose und Behandlung auf wirksame Art und Weise zu kombinieren. - Vermeidung von Infektionen: Entwicklung von robusten Antihaftoberflächen, um eine mikrobielle Besiedlung (die in 5% aller Krankenhausbehandlungen auftritt) effektiv zu vermeiden.
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.
Generation of a mesenchymal niche - Importance of CD34 Research Project | 2 Project MembersDue to its abundance and easy access, subcutaneous adipose tissue has emerged as a potential source of adult stromal/stem cells for tissue engineering and regenerative medicine applications. The stromal vascular fraction (SVF) of adipose tissue indeed includes progenitor cells, referred to as human adipose stromal/stem cells (hASC) with extensive plasticity and the capacity to differentiate into various musculoskeletal lineages. SVF cells express a membrane protein, CD34, in vivo but CD34 expression by hASC spontaneously decreases over time in culture. Culture-expanded hASC are therefore described as CD34 negative cells. CD34 expression in SVF cells has recently been associated with increased biological potential. Preliminary results from our group show that long-term cultures of SVF cells in Petri dishes without passaging into new dishes at cell confluence, in order to favor cell-extracellular matrix and cell-cell interactions, stably maintained and expanded CD34 expression in roughly 20 % of hASC for up to 8 weeks of culture, and that those hASC positive for CD34 expressed higher levels of markers known to be expressed by stem cells. These data suggest the generation of a highly specific hASC culture system in vitro. The ultimate goal of the present project aims at defining the role of CD34 in the biology, function and in vivo potential of hASC. We therefore hypothesize that the expression of CD34 characterizes a subset of hASC with increased self renewal and differentiation capacities. We plan to study the following different aspects: 1. To study the functional role of CD34 in hASC biology. 2. To investigate the exploitability of the hASC obtained without passaging, by testing their in vivo performance in terms of tissue formation, with an emphasis on bone tissue. 3. To analyze the mechanisms involved in the maintenance of CD34-positive cells, i.e. the possible contribution by soluble factors, extracellular matrix and cell-cell interactions. The study proposed here, based on the promising preliminary results already provided by the new protocol to expand hASC, will result in a more in-depth knowledge about the biology and possible self-renewal capacity of hASC, the maintenance of their in vitro environment, and about some molecular players, of which CD34, which could be pivotal in the control of hASC phenotype, differentiation potential and in vivo capability. Thereby, this fundamental study will greatly impact on the applicability of ASC in regenerative medicine in general, and in bone tissue engineering in particular.
Vascularization of tissue engineered grafts Research Project | 2 Project MembersRationale: Vascularization of engineered tissues of clinically relevant sizes is central for cell survival and successful engraftment. In the field of bone regeneration, several studies have established that bone marrow stromal cells, expanded in culture and loaded into porous ceramic scaffolds, are able to form bone tissue, both ectopically and orthotopically. Despite the report of few clinical cases, however, no convincing successes have been achieved in humans, most likely because of lack of sufficient vascular supply, resulting in immediate cell death after implantation. Recent reports in the context of other tissues suggested that addition of vascular cells inside the graft, such as endothelial cells, could improve vascularization and engraftment. Towards this prospective, we have shown that the generation of osteogenic-vasculogenic constructs starting from a single human cell source containing both osteogenic and endothelial cells, namely adipose tissue, is possible. Overall goal: To modulate the relative fractions and level of maturation/organization of human adipose tissue-derived endothelial cell progenitors (EP) and osteoprogenitor cells in a 3D co-culture system and to use the model to investigate the role of a network of EP on the engraftment and bone formation of the resulting engineered construct. Hypothesis: The presence of EP in tissue engineered bone grafts generated by adipose-derived nucleated cells improves and accelerates vascularization of the construct after in vivo implantation, thereby increasing the amount and uniformity of bone tissue formation. Specific aims: 1. Modulate the level of maturation/organization of adipose-derived endothelial cells and osteoprogenitors inside a 3D co-culture system. 2. Investigate the role of an EP network on the vascularization and bone formation of the resulting engineered construct. 3. Investigate the effect of EP in critically sized engineered constructs on cell survival and bone formation in the central core of the grafts. 4. Develop a serum-free medium to support a reproducible and clinically compliant engineering of the defined construct properties. Relevance of the expected results: This project will provide fundamental knowledge on the yet unresolved process by which EP can organize vascular networks and connect to the vasculature of the host in vivo. In the more general field of tissue engineering, it will provide a way to efficiently overcome one of the most critical limitations in current tissue engineering applications, namely vascularization and engraftment of implanted constructs. Finally, this project is very likely to define the starting point for a pilot clinical study using vasculogenic, autologous bone grafts in the treatment of critical size bone defects such as spine fusion or talus reconstruction. A better engraftment, by accelerating the bone healing process, will obviously have a major health and economical significance.
