4:00pm - 4:30pmInvited
Evaluation of the Osteogenic Differentiation of Human and Ovine Mesenchymal Stem Cells (MSC) Using Nanoparticle-based Sensors: MSC-derived Organoid-like as a Model for Tissue Engineering and Bone Disease
1Department of Orthopedics and Trauma Surgery, University Clinic Bonn, Germany; 2Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore; 3Department of Microbiology and Immunobiology, Harvard Medical School, United States; 4Department of Orthodontics, Oral Biology Laboratory, University Clinic Bonn, Germany
Objectives: Bone development and its constant remodeling is a well-regulated biological process orchestrated by MSC-derived osteoblasts forming bone and hematopoietic stem cells derived osteoclasts resorbing bone. MSCs have unique regenerative capacity of the skeleton. Three-dimensional (3D) cell culture technology is one of the fastest growing experimental methods in the life sciences. The 3D cell culture technology has advanced to the degree that is well established for drug discovery and development. The aims of the current study are to investigate and compare the osteogenic differentiation of ovine as a large animal model for orthopedics and human primary MSCs using nanoparticle-based sensors technology.
Methods: Human and ovine MSCs were characterized via their minimal criteria fulfillment and immunomodulatory capacities. Scaffold-free self-organizing microspheres from MSCs were generated on agarose gel. The mineralization of the osteogenic differentiated MSCs was evaluated via standard methods and osteogenic differentiation nanoparticle-derived sensors in 2D and 3D cultures.
Results: Both human and ovine MSCs fulfilled MSCs minimal criteria and showed immunomodulatory capacity by suppressing lymphocytes proliferation. The osteogenic differentiation and mineralization of MSCs was confirmed via specific staining, increased optical density and free phosphate ions. Using specifically developed nanosensors the osteogenic differentiation of both human and ovine MSCs were tracked and visualized in 2D and 3D cell cultures.
Conclusions: We show for the first time real time visualization of human and ovine MSCs osteogenic differentiation in an organoid-like 3D microsphere culture. The organoid-like microtissue can be generated in different sizes and the bioactive environment extracellular matrix (ECM) will be synthesized by the cells themselves mimicking in vivo-like conditions. The organoid-like 3D culture could serve as model for tissue engineering, bone disease modelling and for investigation of ECM target components. The nanoparticle-based sensors offer an enormous opportunity to effectively translate cell therapy from laboratory research into clinical application for regenerative medicine.
4:30pm - 5:00pmInvited
Ice-Templated Biomimetic Materials for Bone Tissue Engineering
Zhejiang University, China
One of the most important issues in bone tissue engineering is searching for new materials and processing techniques to create novel scaffolds with 3-D porous structures. Ideal bone scaffolds should have good biocompatibility, biodegradability, and beneficial mechanical properties. In our research, we use an ice-templating method, attempting to build biomimetic bone implants that adapt to physiological conditions, interact with surrounding tissues, and repair themselves [1-3]. Most recently, we developed a bidirectional freezing technique to achieve hydroxyapatite (HA) scaffold with large-scale aligned porous structure, which could be beneficial for improving cell seeding and migration . By further infiltration such scaffolds with poly (methyl methacrylate) (PMMA), we have fabricated a HA/PMMA composite with nacre-mimetic alternative layered architectures . Such composite has similar composition and mechanical properties with human cortical bone, which make it a promising candidate for bone implants.
 Hao Bai*, Flynn Walsh, Bernd Gludovatz, Benjamin Delattre, Caili Huang, Yuan Chen, Antoni P. Tomsia, and Robert O. Ritchie*, Advanced Materials, 28, 50 (2016).
 Hao Bai*, Yuan Chen, Benjamin Delattre, Antoni P. Tomsia, and Robert O. Ritchie*, Science Advances, 1, e1500849 (2015).
 Hao Bai*, #, Dong Wang#, Benjamin Delattre#, Weiwei Gao, Joel De Coninck, Song Li, and Antoni P. Tomsia, Acta Biomaterialia, 20, 113 (2015).
5:00pm - 5:15pmOral
Study on Effect of Freeze-drying to Decellularized Engineered Hyaline Cartilage Repair Biomaterial
Nanyang Technological University, Singapore
In this study xenologous decellularized biomaterial with hyaline cartilaginous character was developed. To facilitate the transportation of the biomaterial and prepare it for industrialization, the effect of freeze-dry on the biomaterial was studied. This biomaterial has the capacity to repair cartilage damage with a purer collagen type than microfracture. It also has the potential to simplify the current surgical procedure of ACI. The application of xenologous biomaterial avoids the harm to donor site. Hence the elder patients or patients without enough healthy cartilage could be treated. The biomaterial is secreted by chondrocytes encapsulated in 3D culture environment. The cells are co-suspended with gelatin microspheres, which work as porogen, in alginate. After 35 days of proliferation, the secreted extra-cellular matrix is strong enough to hold together the whole graft. Subsequently alginate is removed and a scaffold-free graft is generated. After another 10 days of maturation, the cells are removed by combination of physical, chemical and biological methods. After decellularization, the grafts become scaffold-free acellular xenologous biomaterial. The biomaterial was frozen under -20°C overnight and subsequently freeze-dried. The biomaterial after freeze-drying was immersed in water for one day. The biomaterial experienced the dehydration-rehydration process was compared to the biomaterial without the process (control) in histology and biochemical analysis. Histology results showed very similar morphology of the ECM in H&E staining for both biomaterial with/without dehydration-rehydration. The colour intensity of Safranin O staining and fluorescent intensity of collagen type II was also similar. It suggested the morphology was maintained and the essential biological content including GAG and Collagen II was largely conserved after freeze-drying and rehydration. Biochemical analysis confirmed the results that the total collagen amount had no significant difference between the biomaterial with/without dehydration-rehydration process. This research work further prepared the acellular scaffold-free xenologous biomaterial for industrialization.
5:15pm - 5:30pmOral
Protein Self-Assembly at Oil-Water Interfaces Controls Nanoscale Mechanics, Cell Adhesion and Stem Cell Fate Decision
1School of Engineering and Materials Science, Queen Mary University of London, United Kingdom; 2Institute of Bioengineering, Queen Mary University of London, United Kingdom; 3Department of Chemistry, Umeå University, Sweden
The mechanical behaviour of the extracellular matrix has an important impact on cell phenotype. It has been shown to regulate cell adhesion and spreading, cell motility, proliferation and differentiation in a wide range of cells, stem cells as well as cancer cells. Recently, we reported that keratinocytes and mesenchymal stem cells do not seem to respond to the bulk mechanical properties of silicone materials, in contrast to their behaviour on acrylamide hydrogels. We now show that HaCaT and primary keratinocytes can adhere, spread and proliferate at the surface of non-viscous liquids containing surfactant molecules. We show that the behaviour observed is directly correlated to the concentration of surfactant and that this mediates the assembly and mechanical properties of a nanoscale protein layer at the liquid-liquid interface. We identify that protein self-assembly at the oil-water interface is the main driver of this behaviour. We show that the type and concentration of surfactant as well as the proteins self-assembled control the nanoscale mechanical properties of the resulting interfaces and associated cell behaviour. We show that cell spreading at such liquid interfaces is mediated by integrin ligation, focal adhesion formation and acto-myosin contractility. In addition, we show that this behaviour depends on the interfacial mechanical properties of the protein layer assembled. Finally, we show that keratinocyte differentiation is not initiated by spreading at the surface of liquids, despite the absence of bulk mechanical properties. Our results suggest that nanoscale mechanical properties of biomaterials may dominate over bulk physical properties. This concept has important implications for the design of biomaterials in the field of regenerative medicine.
5:30pm - 5:45pmOral
Molecular Beacon-based Nanosensor for Multiplexed, Continuous Tracking of Intracellular Gene Expression
1School of Chemical & Biomedical Engineering, Nanyang Technological University, Singapore; 2NTU-Northwestern Institute of Nanomedicine, Nanyang Technological University, Singapore
Nanoparticles (NPs) are increasingly utilized to evaluate and optimize cell therapeutics. Existing NP agents have been extensively used to evaluate cell biodistribution following local/systemic administration. Nevertheless, these NPs emit passive signals, unable to provide crucial information regarding cell status post-injection.
Thus far, reporter genes are widely adapted for non-invasive observation of cell behaviors. However, establishing such systems in therapeutic cells is highly inefficient and laborious, accompanied by risk of genomic mutations. Developing a convenient and safe technology to non-invasively examine behaviors of living cells is then a clear priority.
As a solution, we have developed a versatile nanosensor platform capable of probing various cellular functions. The nanosensor is prepared from poly(lactic-co-glycolic acid) NPs which encapsulates and permits sustained delivery of molecular beacon (MB) probes. MBs have high specificity to its complementary target sequence, based on preferential nucleotides matching. Upon successful target hybridization, MBs form a linear structure from its original hairpin, restoring its pre-quenched fluorescence. Therefore, careful design of MB probes allows multiplexed, non-invasive tracking of virtually any mRNA sequence.
Nanosensor performance was firstly validated against ubiquitously-expressed β-actin mRNA. Relative to signal from bolus MBs delivery (Streptolysin-O exotoxin which transiently induce pores), intracellular nanosensor signal lasted significantly (8x) longer. This platform was then applied to track osteogenic & chondrogenic differentiation of mesenchymal stem cells (MSCs). By dynamically monitoring signal ratio between two MBs (targeting differentiation biomarker and housekeeping gene, respectively), we visualized differentiation progression of MSCs toward both lineages. Crucially, observed signal correlated well with quantitative polymerase chain reaction analysis. Moreover, nanosensor labeling did not significantly influence the differentiation processes.
In conclusion, a safe, versatile nanosensor platform for non-invasive cell tracking is described herewith. Given its compatibility towards multiplexed evaluation even in complex culture systems, we anticipate such nanosensor to partake a crucial role in optimizing cell-based therapeutics.