Projects & Collaborations 2 foundShow per page10 10 20 50 A 3D microfluidic osteochondral model to investigate mechanisms triggering osteoarthritis and therapeutic effects of bioactive factors produced by human nasal chondrocytes Research Project | 1 Project MembersOsteoarthritis (OA) is a multifactorial disorder affecting up to 15% of the adult population and involving the joint as a whole. The different therapeutic options proposed are predominately palliative and still far from being effective in reversing the pathology. We recently demonstrated that nasal chondrocytes (NC) can be used clinically for repair of traumatic articular cartilage defects in the knee. The possibility to extend indications of NC to stages of early osteoarthritis would represent a step forward in this area of research. The development of reliable OA in vitro models is thus of primary importance to advance the research into causes and to design potential therapeutics. Different innovative approaches have been proposed, relying on the generation of 3D cartilage/osteochondral constructs obtained through biomaterials and/or bioreactor. However, integrating all key components of OA environment in a single system still remains an open challenge. Working hypothesis. In the proposed research, we will use a microfluidic hydrogel platform to generate physiological and pathological models of osteochondral units. The model will be first exploited to elucidate mechanisms of cartilage/bone cross talk during the triggering of OA, and subsequently to screen novel OA disease-modifying compounds. Specifically, we hypothesize that factors released by nasal chondrocytes are potential novel candidates as OA disease-modifying compounds.Specific aims. We will first address the generation of a 3D microscale hydrogel model of a human osteochondral (OC) unit, consisting of a cartilaginous layer integrated with an endothelialized bone layer (Aim1). We will then switch the OC healthy model towards OA phenotype, by perturbing it through inflammatory treatments, pathological/physiological-like mechanical loadings and their combination (Aim2). The OA model will be then used to investigate the main molecular pathways involved in the cross talk between cartilage and bone cells during the triggering of the pathology (Aim3). Finally, we will exploit the generated model to test the anti-OA effect of factors released by NC, based on their capacity to reverse OA like traits (Aim4).Experimental design. A microscale platform will be developed, comprising a 3D spatially organized co-culture module, equipped with an integrated system for biochemical and mechanical stimulation. A healthy model of OC unit will be first established, consisting of a cartilaginous layer (generated by human articular chondrocytes, AC) integrated with a bone layer (generated by human mesenchymal stem/stromal cells - MSC - or osteoblasts). The feasibility of AC and MSC/osteoblasts to differentiate into phenotypically stable and integrated cartilage-bone composites and the ingrowth of a vascular-like structure within the bone layer will be evaluated. To switch from healthy to OA-like phenotype, the OC unit will be exposed to inflammatory factors (IL-1ß, TNF-a, IFN-?) and/or mechanical loadings. Responses of the system in terms of cartilage/bone matrix degradation, inflammatory and nociceptive markers will be assessed. Bone/cartilage cross talk will be assessed by exposing each layer to conditioned medium collected from the insulted complementary layer, and the role of key molecular pathways will be evaluated (i.e. TGFß/BMP and Wnt/ßCatenin). Finally, factors released by NC will be characterized and used to counteract inflammatory/pain/degrading responses previously induced, together with drugs demonstrated to have anti-inflammatory effects in animal studies. Expected value of the proposed project. Our study will lead to the establishment of the first on-chip in vitro OA model able to recapitulate the key-features of a human osteochondral joint, relevant for gaining insights into OA pathophysiology and anti-OA drug development. In details, thanks to our study we aim at elucidating the role of key molecular pathways in cartilage-bone cross talk during the triggering of OA. Moreover, new insights on the anti-inflammatory and OA-reversing capacity of nasal chondrocytes will be gained, leading to defining putative bioactive factors involved. In turns, this will give new insights for the definition of new therapeutic strategies. 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. 1 1
A 3D microfluidic osteochondral model to investigate mechanisms triggering osteoarthritis and therapeutic effects of bioactive factors produced by human nasal chondrocytes Research Project | 1 Project MembersOsteoarthritis (OA) is a multifactorial disorder affecting up to 15% of the adult population and involving the joint as a whole. The different therapeutic options proposed are predominately palliative and still far from being effective in reversing the pathology. We recently demonstrated that nasal chondrocytes (NC) can be used clinically for repair of traumatic articular cartilage defects in the knee. The possibility to extend indications of NC to stages of early osteoarthritis would represent a step forward in this area of research. The development of reliable OA in vitro models is thus of primary importance to advance the research into causes and to design potential therapeutics. Different innovative approaches have been proposed, relying on the generation of 3D cartilage/osteochondral constructs obtained through biomaterials and/or bioreactor. However, integrating all key components of OA environment in a single system still remains an open challenge. Working hypothesis. In the proposed research, we will use a microfluidic hydrogel platform to generate physiological and pathological models of osteochondral units. The model will be first exploited to elucidate mechanisms of cartilage/bone cross talk during the triggering of OA, and subsequently to screen novel OA disease-modifying compounds. Specifically, we hypothesize that factors released by nasal chondrocytes are potential novel candidates as OA disease-modifying compounds.Specific aims. We will first address the generation of a 3D microscale hydrogel model of a human osteochondral (OC) unit, consisting of a cartilaginous layer integrated with an endothelialized bone layer (Aim1). We will then switch the OC healthy model towards OA phenotype, by perturbing it through inflammatory treatments, pathological/physiological-like mechanical loadings and their combination (Aim2). The OA model will be then used to investigate the main molecular pathways involved in the cross talk between cartilage and bone cells during the triggering of the pathology (Aim3). Finally, we will exploit the generated model to test the anti-OA effect of factors released by NC, based on their capacity to reverse OA like traits (Aim4).Experimental design. A microscale platform will be developed, comprising a 3D spatially organized co-culture module, equipped with an integrated system for biochemical and mechanical stimulation. A healthy model of OC unit will be first established, consisting of a cartilaginous layer (generated by human articular chondrocytes, AC) integrated with a bone layer (generated by human mesenchymal stem/stromal cells - MSC - or osteoblasts). The feasibility of AC and MSC/osteoblasts to differentiate into phenotypically stable and integrated cartilage-bone composites and the ingrowth of a vascular-like structure within the bone layer will be evaluated. To switch from healthy to OA-like phenotype, the OC unit will be exposed to inflammatory factors (IL-1ß, TNF-a, IFN-?) and/or mechanical loadings. Responses of the system in terms of cartilage/bone matrix degradation, inflammatory and nociceptive markers will be assessed. Bone/cartilage cross talk will be assessed by exposing each layer to conditioned medium collected from the insulted complementary layer, and the role of key molecular pathways will be evaluated (i.e. TGFß/BMP and Wnt/ßCatenin). Finally, factors released by NC will be characterized and used to counteract inflammatory/pain/degrading responses previously induced, together with drugs demonstrated to have anti-inflammatory effects in animal studies. Expected value of the proposed project. Our study will lead to the establishment of the first on-chip in vitro OA model able to recapitulate the key-features of a human osteochondral joint, relevant for gaining insights into OA pathophysiology and anti-OA drug development. In details, thanks to our study we aim at elucidating the role of key molecular pathways in cartilage-bone cross talk during the triggering of OA. Moreover, new insights on the anti-inflammatory and OA-reversing capacity of nasal chondrocytes will be gained, leading to defining putative bioactive factors involved. In turns, this will give new insights for the definition of new therapeutic strategies.
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.
