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Microfluidic assay of FGF2 therapeutic administration for bone regeneration

Microfluidic assay of FGF2 therapeutic administration for bone regeneration 2017-2019
Acronym: μFGF2bone
Project director: Gabriela Chiritoiu

Musculoskeletal disorders affect 1 in 7 people and fractures alone affect 1 in 50 people annually while 10% of bone injuries fail to heal. Our present proposal aims to test for the first time the potential of fibroblast growth factor-2 (FGF2), to be administered as a stimulatory drug to enhance bone regeneration.

Musculoskeletal disorders affect 1 in 7 people and fractures alone affect 1 in 50 people annually while 10% of bone injuries fail to heal. Our present proposal aims to test for the first time the potential of fibroblast growth factor-2 (FGF2), to be administered as a stimulatory drug to enhance bone regeneration.

Our interdisciplinary approach includes identifying potential positive effect of FGF2 on osteogenic differentiation in an osteo-adipo-inductive environment and screening of specific dosage and temporal administration protocols by using a microfluidic device to control drug perfusion frequency. The innovation of the microfluidic chip will consist of the proposed design and fabrication of channel configuration onto glass surfaces. The developed chip will be connected to a pressure-based flow controller in order to achieve a fluid flow through the micro-channels.

The main deliverables of our research and experimental development activities are:

  • development of specific lab-on-a-chip devices with capacities of providing chemical stimuli for cellular osteogenic growth
  • development of specific dosage and temporal administration protocol of FGF2 for efficient enhancement of osteogenic differentiation of mesenchymal stem cells.

At the end of the project, our laboratory technological development will generate a proposed schema of administration of the investigated drug using a microfluidic-based approach. This can be used in the future as a platform to screen drugs for bone diseases and to study their mechanism of action.

The project μFGF2-bone has three main Objectives as follows:

  • To characterize the FGF2 effect on MSCs differentiation to osteoblasts, under concomitant exposure to pro-adipogenic and pro-osteogenic conditions. The existing preliminary data will be reproduced in concomitent osteoinduction and adipoinduction stimuli exposure with a view to precisely fine-tune the exact time window of sensitivity of MSCs to FGF2 in a more relevant environment. The experimental set-up is already established, therefore we expect to efficiently perform the necessary experiments using a mixture of osteogenic and adipogenic media instead of osteogenic factors alone. ;
  • To design, fabricate and validate a microfluidic device for controlled FGF2 release. The steps for fabrication of microfluidic chips for cell assays have already been performed by Dr. F. Sima, partner 1 team leader. Validation and characterisation of fluid flow through the channels having different architectures will be performed by time-lapse imaging techniques. We expect the perfused FGF2-containing cell culture media to change gradient velocity through the sinusoidal microchannels as compared to linear ones. This activity will be performed regularly for each created microdevice before use for MSC culture.
  • To investigate the potential of FGF2 to stimulate bone regeneration. For potential therapeutic schemes, FGF2 will be in vitro evaluated for stimulating bone tissue formation.
Project Leader Gabriele Chiritoiu, PhD - IBAR
Project Supervizer Petrescu Stefana, PhD - IBAR
PhD student Laura Georgiana Manica - IBAR
Head P1 Sima Felix - INFLPR
Researcher Axente Emanuel - INFLPR
Researcher Jipa Florin - INFLPR
PhD Student Calin Bogdan Stefanita - INFLPR
Research Assistant Stefana Iosub - INFLPR

The main results of the project leading to the development of an original lab-on-a-chip device are the following:

2017

  • Biochip design and tests. We designed a 4 reservoirs T shaped microchip able to generate a gradient of the administered drug and favor cell cultivation. The design led to the production of a first microfluidic glass platform was produced using advanced laser technologies and thermal treatment.
  • Real time imaging of cells cultivated biochip under FGF2 treatment. Using a CytoSmart device we were able to document the differentiation of mesenchymal stem cells to osteoblasts by FGF2 administration within the biochip in real time.
  • Analysis of cell differentiation during FGF2 treatment by mineralization, identification of differentiation markers and assay of MAPK activation. The detailed analysis of the mesenchymal stem cells differentiation using three different biochemical assays revealed that cells cultivated in the biochip in the presence of FGF2 are able to efficiently differentiate to osteoblasts. The data revealed the parameters of the biochip required for its efficient functioning.

2018

  • Generation of the lab-on a chip device. We have developed a specific lab-on-a-chip device with capacities of providing chemical stimuli for cellular osteogenic growth, as shown below.

  • Protocol for FGF2 treatment with the biochip. Further, we have generated a protocol for the specific dosage and temporal administration of FGF2 for efficient enhancement of MSCs osteogenic differentiation. As seen below, the protocol successfully resulted in the differentiation of stem cells to osteoblasts( Alizarin red staining).

     
Our microfluidic-based approach can be used in the future as a platform to screen drugs for bone diseases and to study their mechanism of action. This has a significant impact as a platform of in vitro drug screening that would decrease the need of pre-clinical tests on animals and accelerate development of new therapies.

 

Articles

Emanuel Axente and Felix Sima, "Biomimetic nanostructures with compositional gradient grown by combinatorial matrix-assisted pulsed laser evaporation for tissue engineering". Current Medicinal Chemistry,2018 -in evaluation

F. Jipa, S. Iosub, B. Calin, E. Axente, F. Sima, K. Sugioka, "High repetition rate UV versus VIS picosecond laser for fabrication of 3D microfluidic channelsembedded in photosensitive glass". Applied Surface Science-2018- to be submitted

 

Conferences

Photon-assisted synthesis and processing of materials in nano-microscale, Strasbourg, France, 2018, Oral Presentation "3D embedded structures fabrication in photosensitive glasses by high-repetition-rate picosecond laser", Jipa Florin

Laser Ignition Summer School, 2018, Poster "Microfluidic device fabrication in photosensitive glasses by high repetition rate picosecond lasers, Florin Jipa, Stefana Iosub, Emanuel Axente, Bogdan Calin, Koji Sugioka, Felix Sima