Utilizing Tissue Clearing Based 3-D Imaging to Quantitatively Study Neural Regulation of Craniofacial Mesenchymal Stem Cells Grant uri icon


  • Utilizing tissue clearing based 3-D imaging to quantitatively study neural regulation of craniofacial mesenchymal stem cellsRecent discoveries of bone regulation by the nervous system has led to the identification of newregulatory axes between the nervous system and bone1-3. Various studies indicate that signalfrom the central or peripheral nervous system directly or indirectly regulates the bone remodelingprocess1, 2.One technical challenge on studying the nerve-bone interactions is to visualize nerve fibers on orwithin bones because of their fine size (~1µm diameter) and the distant location of the neuron cellbodies. In addition, 2-D information obtained from sections made it difficult or impossible toquantify spatial interactions between complex craniofacial tissues. Development of 3-D imagingbased tissue clearing technique provides solutions to these two issues. Tissue clearing techniqueis a recent breakthrough in the neuroscience field. By rendering tissue transparent, 3-D imagesof the whole organ can be achieved directly with a confocal microscope without sectioning4.We recently developed a new tissue clearing technique named PEG Associated SolventSystem (PEGASOS). The PEGASOS method efficiently clears both hard and soft tissue to hightransparency and protects endogenous fluorescence with no loss. The PEGASOS method wasinitially developed to study the neuron connectome within the brain (manuscript in revision). Pilotexperiments suggested that it can also be applied on the craniofacial hard tissue. Skull bone,teeth and femur harvested from adult mice can be cleared to nearly invisible after treatmentwithout losing GFP fluorescence. We were able to acquire the 3-D images of vascular networkwithin the tooth, suture, dura and calvarial bone. We were also able to acquire the 3-D images ofnerves within the femur periosteum, tibia bone marrow, suture and periodontal ligamental space.Quantitative analysis of these 3-D images indicated that vasculatures and nerves withincraniofacial hard tissue present highly heterogenous distribution pattern. In contrast to previousstudy, our preliminary results suggested that Gli1+ cells within the suture are closely associatedwith the vasculatures.In the current proposal, we propose to improve the PEGASOS method for craniofacial hardtissue research. With the spatial information provided by PEGASOS based 3-D imaging,we would like to quantitatively test the hypothesis that craniofacial MSCs are associatedwith the neurovascular bundle (NVB) and nerves are essential for stem cell migrationtowards the injury site

date/time interval

  • 2018 - 2020