Periodic Reporting for period 4 - COLMIN (A Google Earth Approach to Understanding Collagen Mineralization)
Berichtszeitraum: 2022-08-01 bis 2024-01-31
Although with our aging population bone related diseases become a more and more import problem in healthcare, there is still relatively little known about the mechanisms underlying bone formation.
The focus of COLMIN is to understand the mechanisms of collagen mineralization in bone, where the growth of inorganic apatite is directed by the dynamic interaction with collagen, non-collagenous proteins, and carbohydrates over different hierarchical levels, from the nanometer to the multimicron scale. In addition, we investigate pathological mineralization processes in bone diseases. Understanding these processes not only builds our fundamental understanding of bone formation but is also crucial for the development of treatments for bone defects and mineralization-related diseases.
We use both top-down (tissues), and bottom-up (cell culture) approaches to investigate the chemistry and structure of the extracellular matrix. To capture the mineral formation processes' dynamics, we use 2D and 3D in vitro model systems - including bone-on-chip systems and genetically engineered stem cells - aiming for live imaging with nanoscale resolution. To unravel the detailed ultrastructure of the mineralized/affected tissues, we use advanced electron microscopy and fluorescence imaging as well as spectroscopy.
To achieve our goals, we bring together many disciplines, including biochemistry, materials chemistry, spectroscopy, and advanced electron microscopy. Our research also involves collaborations with (inter)national top experts in various areas, with companies to push the limits of our imaging capabilities, and with clinicians to apply our knowledge in future patient care.
The focus of COLMIN is to understand the mechanisms of collagen mineralization in bone, where the growth of inorganic apatite is directed by the dynamic interaction with collagen, non-collagenous proteins, and carbohydrates over different hierarchical levels, from the nanometer to the multimicron scale. In addition, we investigate pathological mineralization processes in bone diseases. Understanding these processes not only builds our fundamental understanding of bone formation but is also crucial for the development of treatments for bone defects and mineralization-related diseases.
We use both top-down (tissues), and bottom-up (cell culture) approaches to investigate the chemistry and structure of the extracellular matrix. To capture the mineral formation processes' dynamics, we use 2D and 3D in vitro model systems - including bone-on-chip systems and genetically engineered stem cells - aiming for live imaging with nanoscale resolution. To unravel the detailed ultrastructure of the mineralized/affected tissues, we use advanced electron microscopy and fluorescence imaging as well as spectroscopy.
To achieve our goals, we bring together many disciplines, including biochemistry, materials chemistry, spectroscopy, and advanced electron microscopy. Our research also involves collaborations with (inter)national top experts in various areas, with companies to push the limits of our imaging capabilities, and with clinicians to apply our knowledge in future patient care.
A living In vitro system to dynamically study collagen mineralization
The mineralization of collagen in bone is one of the most crucial processes in our body as it supplies the skeleton on which we depend for support and protection. Although collagen mineralization has been studied for decades, still major knowledge gaps exist with regards to the molecular mechanisms of the process.
The most advanced model for new (woven) bone formation
Due to dynamics and compositional complexity, in vivo studies cannot give a comprehensive picture of collagen mineralization, while non-living, biomimetic in vitro studies do not fully represent the in vivo system. To close the gap between in vivo and in vitro strategies we generated the first bone organoid: a self-organizing 3D tissue culture (a living in vitro system) that produces an extracellular matrix with all main characteristics of bone. It consists of human mesenchymal stem cells that differentiate into an organized co-culture of osteoblasts and osteocytes. These cells communicate by signaling proteins, and become embedded in a collagenous matrix that containing the key non-collagenous molecules and which is becomes mineralized under biological control.
A Google Earth imaging workflow.
To study the dynamics of the osteogenic process at all required length scales, we designed a strategy in which live fluorescence imaging is coupled through direct high pressure freezing to cryo-fluorescence, cryo-Raman and 3D cryo electron microscopy. Together with two companies, Zeiss and CryoCapCell, we developed a unique workflow to realize this. During (3D) live fluorescence imaging of cell cultures the event of interest is captured by cryo-arrest using high pressure freezing. The samples can then be transferred to be imaged with 3D cryo-fluorescence microscopy to precisely define the region of interest, and with preservation of the coordinates, be transferred to cryoFIB/SEM. There, the coordinates guide us to the selected area to be imaged with a voxel size of ~5 nm.
The correlation of 3D Raman microscopy with SEM, on the other hand, allows to overlay chemical and structural information on bone matrix composition revealing novel chemical aspects in the anomalies that are observed in genetic bone diseases.
With the ultimate aim of achieving live imaging with nanometer resolution, we are developing methodology for liquid phase electron microscopy. To maximize contrast and to minimize damage we have been collaborating with two companies, Denssolutions and VitroTEM, on the development of graphene-based liquid cells. With Denssolutions we have achieved graphene coated liquid cells that we successfully used in the first trials. With VitroTEM we have successfully achieved the automated encapsulation of collagen samples in large liquid-filled graphene pockets on regular TEM grids. Of even greater importance is the successful application - together with consortium members from Leiden and Amsterdam – for a National Facility for Liquid phase electron microscopy of biological materials, position in the Radboudumc electron microscopy center, which will house the world’s first electron microscope specifically configured for this purpose.
Understanding the role of non-collagenous proteins in collagen mineralization.
Parallel to our efforts in visualizing the presence and action of non-collagenous proteins (NCPs) in collagen mineralization, we have performed in vitro studies in the absence of NCPs. These studies reveled that not NCPs but nm-sized tubular channels inside the collagen fibrils are responsible for directing the crystallographic orientation of the intrafibrillar crystals. Our latest results show that using only a biomimetic mineralization solution with polyaspartic acid as a mineralization control agents, we can reconstitute the mineralized matrix of previously demineralized bone. We achieved the successful reconstitution of the bone structure from the level of the mineralized fibril, all the way to the level of the osteon.
The mineralization of collagen in bone is one of the most crucial processes in our body as it supplies the skeleton on which we depend for support and protection. Although collagen mineralization has been studied for decades, still major knowledge gaps exist with regards to the molecular mechanisms of the process.
The most advanced model for new (woven) bone formation
Due to dynamics and compositional complexity, in vivo studies cannot give a comprehensive picture of collagen mineralization, while non-living, biomimetic in vitro studies do not fully represent the in vivo system. To close the gap between in vivo and in vitro strategies we generated the first bone organoid: a self-organizing 3D tissue culture (a living in vitro system) that produces an extracellular matrix with all main characteristics of bone. It consists of human mesenchymal stem cells that differentiate into an organized co-culture of osteoblasts and osteocytes. These cells communicate by signaling proteins, and become embedded in a collagenous matrix that containing the key non-collagenous molecules and which is becomes mineralized under biological control.
A Google Earth imaging workflow.
To study the dynamics of the osteogenic process at all required length scales, we designed a strategy in which live fluorescence imaging is coupled through direct high pressure freezing to cryo-fluorescence, cryo-Raman and 3D cryo electron microscopy. Together with two companies, Zeiss and CryoCapCell, we developed a unique workflow to realize this. During (3D) live fluorescence imaging of cell cultures the event of interest is captured by cryo-arrest using high pressure freezing. The samples can then be transferred to be imaged with 3D cryo-fluorescence microscopy to precisely define the region of interest, and with preservation of the coordinates, be transferred to cryoFIB/SEM. There, the coordinates guide us to the selected area to be imaged with a voxel size of ~5 nm.
The correlation of 3D Raman microscopy with SEM, on the other hand, allows to overlay chemical and structural information on bone matrix composition revealing novel chemical aspects in the anomalies that are observed in genetic bone diseases.
With the ultimate aim of achieving live imaging with nanometer resolution, we are developing methodology for liquid phase electron microscopy. To maximize contrast and to minimize damage we have been collaborating with two companies, Denssolutions and VitroTEM, on the development of graphene-based liquid cells. With Denssolutions we have achieved graphene coated liquid cells that we successfully used in the first trials. With VitroTEM we have successfully achieved the automated encapsulation of collagen samples in large liquid-filled graphene pockets on regular TEM grids. Of even greater importance is the successful application - together with consortium members from Leiden and Amsterdam – for a National Facility for Liquid phase electron microscopy of biological materials, position in the Radboudumc electron microscopy center, which will house the world’s first electron microscope specifically configured for this purpose.
Understanding the role of non-collagenous proteins in collagen mineralization.
Parallel to our efforts in visualizing the presence and action of non-collagenous proteins (NCPs) in collagen mineralization, we have performed in vitro studies in the absence of NCPs. These studies reveled that not NCPs but nm-sized tubular channels inside the collagen fibrils are responsible for directing the crystallographic orientation of the intrafibrillar crystals. Our latest results show that using only a biomimetic mineralization solution with polyaspartic acid as a mineralization control agents, we can reconstitute the mineralized matrix of previously demineralized bone. We achieved the successful reconstitution of the bone structure from the level of the mineralized fibril, all the way to the level of the osteon.
So far, the project has largely focussed on establishing the methodology required for the excecution of the project. The availability of these tools will now allow us to apply them to investigate the mechanisms of collagen mineralization.
Our bone organoid was the first organoid for new (woven) bone and will allow us to study bone formation processes in a living system with unprecedented detail. It also allows us to expand the approach to patient material by the use of induced pluripotent stem cells (iPSCs) or through the de-differentiation to stem cells of patient (skin) fibroblasts.
The 3D live-to-cryo imaging workflow we developed is unique and will allow us to investigate the finest details of collagen assembly and mineralization processes at any point in space or time. Of particular importance here will be the combination of microscopy, with is now available through the combined use of Raman microscopy, mass spectrometry and imaging, and in the coming period through the development of a dedicated cryoFIB/SEM-SIMS instrument with Orsay-Physics.
The acquisition of a dedicated TEM for LP-EM together with the development of graphene-based encapsulation technology will now open up the LP-EM field to study biolofgical materials at video rate and with (1-10) nanometer resolution. With this we will realize a totally new window into the study of processes of life with nanometer resolution.
We expect that the use of our cell-culture based systems to study the process of collagen assembly, and the role of glycosylation in directing or preventing mineralization, will provide totally new insights in the mechanisms underlying these processes, and in several cases will force us to correct the accepted current theories.
Our bone organoid was the first organoid for new (woven) bone and will allow us to study bone formation processes in a living system with unprecedented detail. It also allows us to expand the approach to patient material by the use of induced pluripotent stem cells (iPSCs) or through the de-differentiation to stem cells of patient (skin) fibroblasts.
The 3D live-to-cryo imaging workflow we developed is unique and will allow us to investigate the finest details of collagen assembly and mineralization processes at any point in space or time. Of particular importance here will be the combination of microscopy, with is now available through the combined use of Raman microscopy, mass spectrometry and imaging, and in the coming period through the development of a dedicated cryoFIB/SEM-SIMS instrument with Orsay-Physics.
The acquisition of a dedicated TEM for LP-EM together with the development of graphene-based encapsulation technology will now open up the LP-EM field to study biolofgical materials at video rate and with (1-10) nanometer resolution. With this we will realize a totally new window into the study of processes of life with nanometer resolution.
We expect that the use of our cell-culture based systems to study the process of collagen assembly, and the role of glycosylation in directing or preventing mineralization, will provide totally new insights in the mechanisms underlying these processes, and in several cases will force us to correct the accepted current theories.