EXtracellular matrix in CArdiac disease: poLYsaccharides to reBUild the heaRt – EXCALYBUR

Our methodology relies on three complementary technical pillars aimed at creating a biohybrid material capable of mimicking the natural cellular niche.

1. Synthesis and microstructuration of polysaccharide scaffolds We formulated hydrogels based on pullulan and dextran (75:25 ratio), chemically cross-linked with sodium trimetaphosphate (STMP) under alkaline conditions. To generate a network of highly interconnected pores, we optimized a double freeze-drying (2FD) technique. This method yields a more homogeneous and mechanically stable porous structure compared to single freeze-drying. Among the five formulations tested, we selected the PuDColl-2FD scaffold, where collagen type I is introduced via negative pressure pumping after the first freeze-drying cycle to promote cell adhesion.

2. 3D colonization and in situ extracellular matrix (ECM) deposition We seeded Balb/c 3T3 fibroblasts homogeneously throughout the scaffolds using a negative pressure method. Cultures were maintained for 14 days, identified as the ideal compromise to maximize protein secretion while controlling fabrication time. During this period, the cells "decorated" the pores by depositing a matrix rich in collagens I and IV, fibronectin, laminin, and elastin.

3. Innovative decellularization using supercritical CO2 (scCO2) The major innovation of our approach is the use of supercritical CO2 to eliminate cellular components while preserving the deposited matrix. We immersed the scaffolds in a low-concentration SDS solution (0.5%) before subjecting them to an scCO2 cycle at 170 bar and 45 °C for 90 minutes. This process was compared to a standard decellularization method (detergents and enzymes) to validate its superiority in biochemical preservation.

4. Physical, mechanical, and biological characterization We rigorously evaluated our scaffolds at each step. Porosity and morphology: Visualized by scanning electron microscopy (SEM) and 3D confocal imaging. Mechanical properties: Measurement of the shear storage modulus (G’) using vibratory elastography to ensure a stiffness (1.7 to 3.5 kPa) analogous to human soft tissues. Biological efficacy: Quantification of residual DNA via PicoGreen assay and immunostaining of ECM proteins to confirm the integrity of the biological "niche" after treatment.

Validation of a biomimetic and high-performance biohybrid scaffold

1. Physical properties and mechanical stability We validated five hydrogel formulations, identifying the double freeze-drying (2FD) technique as essential for ensuring homogeneous and interconnected porosity. We measured a storage modulus (G’) between 1.7 and 3.5 kPa, placing our scaffolds within the stiffness range of human soft tissues. Furthermore, our results show that the 2FD method provides temperature-independent mechanical stability (25 °C vs 37 °C), unlike single freeze-drying methods.

2. Biological niche optimization and ECM deposition Evaluation of 3D fibroblast culture revealed that coating pores with collagen type I after the first freeze-drying (PuDColl-2FD) is the most effective formulation. In these scaffolds, we observed excellent cell adhesion and proliferation, with 74% to 84% of pores colonized by cells. By day 14 of culture, we confirmed via confocal imaging that cells actively decorate the pores by depositing a complete extracellular matrix (ECM) composed of collagens I and IV, fibronectin, laminin, and elastin.

3. Superiority of supercritical CO2 (scCO2) decellularization The key innovation in our results is the efficacy of scCO2 decellularization. We achieved exceptional purity levels, with less than 1 ng of DNA per mg of scaffold, whereas standard chemical methods leave approximately 55 ng/mg. Most importantly, our imaging and quantification analyses proved that scCO2 preserves the biochemical integrity of the deposited matrix, unlike conventional protocols that lead to a significant loss of essential proteins

Towards a universal platform for regenerative medicine

The convincing results obtained in the EXCALYBUR project open promising horizons for the development of advanced biological substitutes. We have established a solid proof of concept showing that it is possible to create biohybrid materials that combine polymer engineering precision with the richness of cell biology. We envision several areas for development:

1. Adaptation to target tissue specificity One of the major strengths of our strategy is its versatility. We plan to adapt this platform to a wide range of clinical applications by modulating the physicochemical properties of the base scaffold. By using tissue-specific cells, we can generate matrices whose protein composition precisely matches the biological "niche" of the organ to be repaired, such as vascular, hepatic, bone, or neural tissues.

2. Optimization of personalized medicine The cell-deposited matrix (CDM) approach offers immense potential for personalized therapies. By using a patient's own cells to "decorate" our scaffolds before decellularization, we could create custom-made grafts, minimizing immune rejection risks while providing an optimal biochemical environment for regeneration.

3. Clinical transfer and industrialization Scaling up to an industrial level is facilitated by the efficiency of our scCO2 protocol. This rapid process (90 minutes) yields acellular, sterile tissues, essential characteristics for safe clinical application. The mechanical stability of our "off-the-shelf" scaffolds simplifies their storage and handling by surgeons, representing a major economic and logistical advantage.

Heart ischemia (HI) has remained the world's leading cause of death for the past 20 years. Following hypoxia, the heart undergoes several changes developing a chronic disease: heart failure. In recent years, alternative therapies based on tissue engineering have addressed the fundamental problem of losing heart tissue. Recently, the use of the native extracellular matrix (ECM), obtained by decellularization of tissues, has aroused the interest of researchers and clinicians for the repair of the heart. Nevertheless, certain limits linked to the reduced number of donors, the risk of infection and the physical and chemical imbalance within species which can alter the physiology of tissues and cells, must still be overcome. EXCALYBUR aims to create a hybrid material imitating the physical-chemical characteristics and the composition of the cardiac ECM in order to serve as a bioactive scaffold in a HI model. Our hypothesis is that, to meet the requirements of the biomaterial for the repair of the heart, the most adequate approach consists in combining materials with controlled physical-chemical and mechanical properties, with naturally secreted CEM containing the biochemical elements for the tissue repair. This is an innovative approach in which we aim to overcome the limitations of synthetic polysaccharide hydrogels and native ECM, by combining the two concepts in a single biohybrid material. The originality of the project is based on the combination of 100% polysaccharide hydrogels and a method of depositing MEC by cardiac cells to cover the material. This will provide heart cells with an optimized environment compared to other approaches described in the literature, in which one or more components of the ECM are incorporated into the material. As a result, cardiac repair after HI will be promoted by directly using these cell-free patches or these patches for cell therapy, which will lead to improved heart function and tissue repair. An important challenge will consist in developing a method allowing to decellularize the biohybrid material after the deposit of ECM by the cells. To do this, we have already started our collaboration with Dr. P. Subra-Paternault, DR CNRS with 30 years of expertise in the field of supercritical fluids. This technique has been highlighted in recent years to overcome the drawbacks of the more conventional detergeant-based decellularization techniques. To achieve the objective of EXCALIBUR, 3 specific objectives divided into 6 tasks have been defined: I. Development of biohybrid material T1) Characterization of the rat cardiac ECM T2) Formulation of a hydrogel based on polysaccharide having the characteristics of cardiac ECM T3) Coating of the hydrogel with ECM using a manufacturing process by cell secretion II. Decellularization and sterilization of biohybrid material (T4) III. Evaluation of the biohybrid material in vitro and in vivo T5) Loading of the material with cells of the heart muscle and vascular cells and evaluation in vitro T6) Evaluation of the repair of the heart in a model of HI in the rat. The expected results at the completion of the project include: 1) A well-defined map of the most relevant physicochemical properties of cardiac MEC and techniques for characterizing these properties. 2) A method for preparing a cardiac like ECM in three stages: I) synthesis of hydrogel, II) coating of hydrogel with ECM secreted by the cells and III) decellularization. 3) A hybrid material which corresponds to the physical-chemical and biological properties of the native ECM and promotes regeneration in HI.