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Intentional control of mineralized ECM deposition in bone tissue engineering

Final Report Summary - BONECMONITOR (Intentional control of mineralized ECM deposition in bone tissue engineering)

Intentional control of mineralized ECM deposition in bone tissue engineering - the bonECMonitor Project

Summary description of project objectives
Bone tissue engineering has shifted its focus from the attempt to generate functional implants towards the broader understanding of the interplay between the input parameters. Only an increased understanding of how cells react to and interact with biomaterials in specific environments will help us design graft materials that will successfully lead to regeneration of damaged or diseased tissues.
The BonECMonitor project aims at simplifying current bone TE strategies to identify and describe (monitor) the influence of mechanical loading on mineralized ECM development. It contains four work packages (WPs):
• WP1: Development of scaffolds with monodisperse pores
• WP2: Controlled interventions
• WP3: Computational simulation of tissue development
• WP4: Dissemination/outreach activities

In WP1, the existing silk fibroin (SF) scaffolds obtained by the salt leaching method have been modified. Scaffolds with monodisperse pore size distribution and variable pore diameters have been developed by combining existing methods (microfluidic technology and porogen leaching scaffolding technology) and materials in a new way. Human bone marrow derived stromal cells (hMSCs) were able to differentiate into bone forming cells (osteoblasts) and more mineralized ECM was deposited when cells were cultured on the newly developed as compared to salt leached scaffolds. Through inclusion of hydroxyapatite, the scaffolds could be visualized in a physiological environment by µCT.
In WP2, we've found that donor variation and fetal bovine serum (FBS) source influenced cell behavior to a greater than expected extent. FBS batch can be the single determinant of mineralisation in both static and dynamic (spinner flask) condition. We have taken this project even further by investigating the influence of co-culturing hMSC with fluorescently labeled endothelial cells, simulating vasculature. hMSCs seem to be indispensable for endothelial cell support both in 2D and 3D.
We have developed a computational model that simulates the wall shear stresses within scaffolds in a spinner flask bioreactor. This model was able to predict the location of the experimentally formed mineralised tissue both at 60rpm and 300 rpm in WP3.
In WP4, results have been presented at various conferences and two accepted publications and three more in preparation. Further publications have resulted from collaborations that were not funded through this project. The move of the PI to Eindhoven and her capability to attract funding has resulted in various publications such as the BME student association magazine from the TU/e, the Cursor, ETH life, the Annual report from Pirelli 2014, the ICMS Highlights Magazine in 2016 and an interview in the NEMO Kennislink (organisation who aims at bringing research and technology closer to the public, in Dutch). She has further been involved in student supervision, teaching including teaching at winter schools, organizing student workshops or organizing specific sessions of interest at scientific conferences and she was involved in various committee for poster/presentation prizes. She is currently co-organizing the next European Society for Biomaterials Conference in Maastricht (2018) and an active member of the TERMIS EU council and some of the sub-committees.
Main results achieved
• Development of SF scaffolds with monodisperse pore size distribution
• Development of SF scaffolds with increased visibility in µCT in physiological conditions
• Definition of influential mechanical parameters for cell culture studies
• Computational model to predict mineral formation in dynamic bone tissue engineering cultures
• Description of the influence of FBS batch on mineral formation in both static and dynamic bone tissue engineering cultures
• Establishment of a co-culture system of hMSC and endothelial cells in 3D allowing to monitor the formation of vessel-like structures
• Extensive outreach and dissemination activities

Potential impact and use of the project
This project so far provides a very fundamental scientific knowledge concerning bone cells and their interaction with their environment. The results of the bonECMonitor project can be further exploited to study the influence of environmental factors such as mechanical loads, chemical compounds/drugs, structure and material of three-dimensional scaffolds, cell source (healthy or diseased donors) on tissue-engineered bone formation in a 3D environment and provide input data for computational simulations. It is expected to increase our current understanding of the interplay between parameters in a substantial way. Such an understanding may - in the long term - enable to design innovative bone grafts that promote regeneration better than current solution because they are based on our understanding of what the specific patient's needs are.
In addition, this project has aided the PI's integration in her current research environment, by giving her the opportunity to establish this research field in her new environment and has aided her promotion to a permanent position. It has increased the PI's personal network through new collaborations, increased her visibility and improved her recognition as an independent leader in the bone tissue engineering field.