INTERFACE

Tissues and cell Interfaces in muscle-skeleton system: Application to the design of bioartificial SoS 

Principal investigator : Cécile Legallais (BMBI)

Context and scientific approach

Fig. 1 : Multi-scale and multi-components approaches of the muscle-tendon-bone system, elbow shown as an example.

Since 15-20 years, the achievements in biotechnology have fostered significant progresses in the field of tissue engineering. Tissue engineering aims at favoring in situ tissue regeneration or at building functional tissue substitutes that could be implanted to replace injured or pathological ones. This emerging science gathers interdisciplinary skills at different scales (engineering sciences such as biomechanics, materials science, instrumentation, biology, physics and chemistry, clinical sciences). Lab design of some engineered tissues has already led to clinical application of products that are now defined as ATMP (advanced therapy medicinal products). There are numerous examples for single biohybrid tissues such as bone, skin, muscle, cartilage, liver... In any case, tissue engineering integrates cells into a scaffold where they can attach, proliferate or differentiate. In addition, the use of bioreactor is requested to perform 3D culture and mimic the cells’ in vivo niche and environment, while ensuring a better control of cell culture condition and possibly induce cell response to mechanical stimuli.

Our achievements at UTC, as well as discussions with our partners “end-users” involved in maxillofacial or orthopedic surgery led us now to raise a new “challenge”: to target global system development in which different multi-layered tissues will be designed together. This concept supposes to study the interfaces between the biohybrid implant and the surrounding tissues, especially from conjonctive or muscle origin, for an application in maxillofacial surgery, in dental implants, or any other cases of repair for the muscle-tendon-bone continuum (Fig. 1). It represents an example of complex bioinspired system of systems in which the different systems are in continuous changes and interactions. The structural complexity (reconstructed multi-layered tissue) of the proposed bioengineered systems that makes them versatile also presents a major challenge to understand and predict their mechanical and biological behavior.

Objectives

Fig. 2: Schematic representation of the Challenge “Interface”. The overall purpose is to reconstruct the continuum bone-tendon-muscle using smart tissue engineering approaches based on i) a strong knowledge of the native tissues architecture and composition at different scales, ii) the design of electrospun materials mimicking the structure, iii) the use of relevant cell types, iv) the culture of the biohybrid constructs under dynamic conditions in bioreactors to exploit mechanical stimulation (as in a body) for enhanced tissue maturation.

In this challenge, we propose an overall methodology to design and validate a bioartificial system representing the continuum muscle-tendon-bone, itself composed of biohybrid systems at different scales (Fig. 2).

In this framework, the interactions involved between the different systems are contingent to the biological properties of the developed tissues, the chemical and mechanical properties of the used material scaffold. These interactions need to be monitored through the culture process at the relevant length scale to first tune the different cells response toward the desired type of tissue and tailor the mechanical properties of the global system of systems.

The scientific approach consists in putting together knowledge regarding the native musculo-skeletal at different scale to guide tissue engineering development.

Permanent researchers and staff involved in the project:

Fahmi Bedoui (MCF HDR at Roberval lab, UMR CNRS 7337)
Sabine Bensamoun (CR1 CNRS at BMBI lab, UMR CNRS 7338)
Jean-François Grosset (MCF at BMBI lab, UMR CNRS 7338)
Murielle Dufresne (PRAG at BMBI lab, UMR CNRS 7338)
Christophe Egles (Professor at BMBI lab, UMR CNRS 7338)
Patrick Paullier (IE CNRS, BMBI lab, UMR CNRS 7338)
Quentin Dermigny (AI, BMBI lab, UMR CNRS 7338).

Collaborations:

Service de Chirurgie Maxillo-Faciale, Amiens University Hospital (Prof. B. Devauchelle, Prof. S. Testelin)
Université de Picardie Jules Verne, EA 4666 (Dr S. Le Ricousse, Dr. M. Naudot)
Institut de Biologie Paris Seine (UPMC-INSERM-CNRS) (Dr. D. Duprez, DR CNRS, L. Gaut, PhD)
ICEPEES Strasbourg (Prof. G. Schlatter, Dr. A. Hebraud)
Institute of Sport Medicine, Copenhagen, Denmark (Prof. P. Magnusson)
Institute of Multiphase Processes, Leibniz University, Hannover, Germany (Prof. B. Glasmacher)
Mayo Clinic, Rochester, USA.

Post-doctoral positions:

Malek KAMMOUN (Feb 2017 – Feb 2019, Labex MS2T funding)
Firas FAHRAT (July 2017 – June 2018, Région Hauts de France funding)

PhD students:

Alejandro GARCIA GARCIA (Oct 2015 – Oct 2018, Labex MS2T funding)
Megane BELDJILALI-LABRO (Oct 2016 – Oct 2019, Labex MS2T funding)

Master students:

Moritz von Wrangler (April 2016 – September 2016, ERASMUS from Leibniz Hannover University, Germany)
Johanna Diekhoff (September 2017 – January 2018, ERASMUS from Leibniz Hannover University, Germany)

Fig. 3. Fluorescence microscopy observation of C3H10T1,2 after 5 days of culture on honeycomb scaffolds. (A) Live and Dead stained cells superposed to Honeycomb. (B) Live and Dead staining on cells. (C) Hoechst 33342 staining all cell nuclei. Calcein AM (green) dye stands for living cells membranes. EthD-1 dye (red dots) stained dead cells nuclei. Scale bar = 100µm.

We first focused on the improvement of the bone biohybrid constructs previously developed at BMBI (Baudequin et al., 2015) and the setting up of parameters for tendon reconstruction, in view of developing the future bone-tendon junction. Specific honeycomb shaped scaffolds were thus prepared by a combined technique of electrospinning and electrospraying by A. Garcia Garcia at the ICEPEES Strasbourg. The hypothesis was that this specific architecture would promote stem cell differentiation into bone lineage. Early osteoinduction was indeed demonstrated with a cell line of mesenchymal stem cells (C3H10) (Fig. 3). We are currently performing in vivo trials in a rat cranial defect model in collaboration with EA4666 in Amiens (Dr M. Naudot).

Figure 4 – Cell culture under mechanical stimuli within the bioreactor T6 CellScale hosting 6 electrospun scaffold cultured with BMSCs. B. BMSCs in static conditions. C. BMSCs under dynamic stretching.

In parallel, several scaffolds were produced at UTC with different types of polycaprolactone (PCL) to guide the same cells to tendon lineage. Some part of the work was most specifically performed in collaboration with the team of D. Duprez (IBPS, UPMC) and was supported by a Convergence Program of Sorbonne Universités. Other scaffolds wee prepared by A. Garcia Garcia at Leibniz University, Hannover, in the framework of a bilateral exchange Grant allocated by the yESAO (European Society for Artificial Organs)., C3H10 cell line was thus cultured on the scaffolds under both static and dynamic conditions for up to 3 weeks. Because the very low extracellular matrix’s synthesis, alterations of the biohybrid construct’s mechanical properties could not be measured. It was thus decided to switch to Bone Marrow Stromal cells (BMSCs) from Spragle Dawly rats and to follow up the culture, so as to favour the neo-synthesis of an extracellular matrix. Cell culture under dynamic stretch shows an alignment of cells following the axis of deformation of the material (Fig.1B), while in static conditions cells remains randomly organized (1C). This aligned morphology reveals that BMSCs are responding to the stress and adapting their phenotype to the mechanical stimuli. In addition, type I collagen fibers (predominant in native tendons) seem to be aligned, mimicking the native structure of tendon.

Fig. 5: AFM experiment to characterize sarcomore’s morphology and elasticitiy

Regarding the muscle and the myotendinous junctions, we led in parallel two approaches: 1) improve the knowledge on this tissue and 2) develop a biohybrid muscle before focusing on the MTJ reconstruction.

A protocol was thus developed by Dr. M. Kammoun (post-doc Labex) to isolate muscle fibers which are stocked in a relaxing solution. Then, under a binocular microscope, single muscle fibers were isolated and submitted to mechanical characterization using atomic force microscopy. AFM technique is based on compressive test using a fine tip (17µm). The challenge was to find a way to fix the muscle fiber during the test to avoid the movements. AFM tests were then run in collaboration with the department of Cellular and physical microbiology of infection located at the Pasteur Institute (Dr Dupres, Lille).

The results showed morphological (size of sarcomere, …) and structural (elasticity, …) properties of the muscle fiber (Fig.5).

For the muscle reconstruction, we chose as first investigation to combine electrospun PCL 10%wt/V (mean fiber diameter 1.2µm +/- 10%) with C2C12 myoblasts After few days of culture, the cell cycle stopped and the cells started to fuse together to form randomly oriented multinucleated myotubes. The unaligned myofibers did not fully matured and did not all gain full contractile function. The levels of C2C12 cell proliferation observed on surfaces coated with laminin were significantly greater than those observed for cells grown on uncoated scaffold and culture plastic.

Fig. 6 : Electrospun scaffolds for muscle tissue engineering. left : unaligned fiber PCL 10% wt/v in 80/20 DCM/DMF; right : Live&Dead cell viability assays, (calcein and propidium iodide) visualization by epifluorescence microscope after 3 days of culture on the scaffold.

Communications in congresses:

• A.GARCIA, G.SCHLATTER, A.HEBRAUD, C.EGLES, F.BEDOUI, C.LEGALLAIS. Bone reconstruction with Polycaprolactone/hydroxyapatite scaffolds. ESB 2016, Lyon, July 2016. Poster.

• A.GARCIA, G.SCHLATTER, A.HEBRAUD, C.EGLES, F.BEDOUI, C.LEGALLAIS. Bone tissue engineering with Polycaprolactone/hydroxyapatite biomimetic scaffolds. 43th ESAO Congress, Warsaw, Sept 2016. Oral presentation.

The cell culture and tissue engineering experiments are conducted on the platform INGESYSBIO at BMBI. Biohybrid constructs are characterized on the platform CARMOD at BMBI and on SAPC electronic microscopy devices.

Three major equipments are more specifically employed: