Periodic Reporting for period 1 - ImmunoBioInks (Instructing Immune System to Regenerate Musculoskeletal Tissues via Structurally Programmable Bio-Inks)
Reporting period: 2021-07-01 to 2023-06-30
Background
The musculoskeletal tissue is the framework of our lives. It holds, shapes and supports freedom of movement of our body and protects the crucial internal organs (brain, heart and lungs). It is responsible for our body’s immunity by providing source of stem cells (bone marrow) that readily transform to immune system cells fighting pathogens, so any damage it poses significant threat to the individual’s quality of life. The patient’s immune system does not only play crucial role in fighting various pathogens but is also vital in inducing normal healing of damaged tissues. Patients, especially with prolonged diseases, ranging from diabetes to HIV tend to have decreasing capacity for healing after injuries due to their compromised immune system.
Goal
The scope of the EU-funded ImmunoBioInks project is to develop 3D-printed materials to treat musculoskeletal defects in patients with an immune system imbalance. The idea is to combine self-assembling peptides, hyaluronic acid (scheme 1) and nanomaterials into printable scaffolds of defined architecture and with carefully designed mechanical properties that can reprogram the patient’s own immune cells. The interaction of immune cells with this innovative 3D scaffold is expected to trigger the necessary healing response.
The musculoskeletal tissue is the framework of our lives. It holds, shapes and supports freedom of movement of our body and protects the crucial internal organs (brain, heart and lungs). It is responsible for our body’s immunity by providing source of stem cells (bone marrow) that readily transform to immune system cells fighting pathogens, so any damage it poses significant threat to the individual’s quality of life. The patient’s immune system does not only play crucial role in fighting various pathogens but is also vital in inducing normal healing of damaged tissues. Patients, especially with prolonged diseases, ranging from diabetes to HIV tend to have decreasing capacity for healing after injuries due to their compromised immune system.
Goal
The scope of the EU-funded ImmunoBioInks project is to develop 3D-printed materials to treat musculoskeletal defects in patients with an immune system imbalance. The idea is to combine self-assembling peptides, hyaluronic acid (scheme 1) and nanomaterials into printable scaffolds of defined architecture and with carefully designed mechanical properties that can reprogram the patient’s own immune cells. The interaction of immune cells with this innovative 3D scaffold is expected to trigger the necessary healing response.
A selection of self-assembling peptides (SAPs) based on the alternation of hydrophobic and hydrophilic amino acids: DABACABACD (A: hydrophobic residue: F phenylalanine or Y tyrosine, B/C: hydrophilic residue e.g.: K lysine or E glutamic acid) was designed. A parametric study was then carried out to verify the effect of rational peptide sequence modification on the final physicochemical properties of hydrogels. Tyramine-modified hyaluronan (THA) was also synthesized at two different molecular weights (280 kDa and 1640 kDa) and used to form composite hydrogels with self-assembling peptides. All parental peptides self-assembled into semi-flexible networks and hydrogels above critical gelation concentration in the region of 2.5-5 mM and display characteristic high β-sheet content. Self-assembly, rheological properties and printability of both peptide and peptide-THA hydrogels can be controlled by the choice of primary peptide sequence, fabrication technique and final crosslinking mechanisms including enzymatic (horseradish peroxidase, H2O2) and visible green light crosslinking using Eosin. These hydrogels are characterized by shear-thinning behavior and rapid recovery allowing extrusion-based fabrication of scaffolds. For the first time we also demonstrated the polarization effects of the supplemented THA on macrophages differentiated from human peripheral blood mononuclear cells over 5 days. M1 and M2 polarization modulated by the supplementation with low and high molecular weight THA were unraveled by the semi-automated image analysis from confocal microscopy, gene expression analysis and ELISA. Self-assembly, rheological properties and printability of SAPs were controlled by the choice of primary peptide sequence, concentration, and fabrication technique. Furthermore, the position of tyrosine in the peptide sequences dictated the distinct self-assembly into nanostructures, with sequences EF8 (control), EF8Y, YEF8Y, EYF8 self-assembling into thin fibers d<4 nm, whereas YEF8 self-assembling into rod-like flat ribbons d>20 nm. These distinct structural changes influenced the inflammatory profile, with peptides EF8 and EYF8 displaying anti-inflammatory profile, peptides EF8Y, YEF8Y remaining inert and peptide YEF8 displaying a pro-inflammatory state. In summary we uncover the link between basic molecular interactions driving self-assembly of tyrosine-containing SAPs into distinct nanostructures and demonstrate them as extrudable platforms for immunomodulatory tissue engineering. Finally, using traditional simplistic approach of “M1-like” as pro-inflammatory, and “M2-like” to as anti-inflammatory states, respectively, we confirm the typical association of low versus high molecular weight for chemically modified THA holds true and we thus bring immunomodulatory guidance towards the appropriate choice of it to a wider biomaterials and tissue engineering communities. We believe that these responses provide more rapid and accurate in vivo representation and will provide translational basis for the use of THA-based biomaterials in the future.
One important aspect of getting biomaterials towards clinically viable products is understanding how sterilization affects its properties. During the action, we have contacted a company focussing on that, Sterigenics, to start a collaborative project evaluating the effects of various sterilization methods of our developed biomaterials. Unfortunately, the company has withdrawn their interests in our project. Nevertheless, during the project with our partners we have evaluated sterilization effects on a branch of biomaterials as noted above.
The immunomodulatory properties of hyaluronan and its derivatives as well as peptides are key to their use in medicine and tissue engineering. In our work we evaluated the capability of soluble tyramine-modified hyaluronan (THA) of two molecular weights (low Mw=280 kDa and high Mw=1640 kDa) for polarization of peripheral blood mononuclear cells-derived macrophages. We confirmed the typical association of low versus high molecular weight for THA holds true and stipulated that these responses will provide more accurate in vivo representation and translational immunomodulatory guidance for the use of THA-based biomaterials to a wider biomaterials and tissue engineering communities, providing a benchmark for the future clinical use of these biomaterials. Self-assembly, rheological properties, and printability of peptide hydrogels can be controlled by the choice of primary peptide sequence. This knowledge will help in the future to rationally design peptide hydrogels for applications in regenerative medicine, as well as provide immunomodulatory understanding of influence of nanostructured peptides and proteins. The overall immunomodulatory rules will have an impact to the clinically relevant scenarios, and this has been discussed with Spatha Medical company (based in France), representative of which has been invited to a technological symposium organized by fellow on European Society of Biomaterials (ESB) 2022 conference held in Bordeaux.
The immunomodulatory properties of hyaluronan and its derivatives as well as peptides are key to their use in medicine and tissue engineering. In our work we evaluated the capability of soluble tyramine-modified hyaluronan (THA) of two molecular weights (low Mw=280 kDa and high Mw=1640 kDa) for polarization of peripheral blood mononuclear cells-derived macrophages. We confirmed the typical association of low versus high molecular weight for THA holds true and stipulated that these responses will provide more accurate in vivo representation and translational immunomodulatory guidance for the use of THA-based biomaterials to a wider biomaterials and tissue engineering communities, providing a benchmark for the future clinical use of these biomaterials. Self-assembly, rheological properties, and printability of peptide hydrogels can be controlled by the choice of primary peptide sequence. This knowledge will help in the future to rationally design peptide hydrogels for applications in regenerative medicine, as well as provide immunomodulatory understanding of influence of nanostructured peptides and proteins. The overall immunomodulatory rules will have an impact to the clinically relevant scenarios, and this has been discussed with Spatha Medical company (based in France), representative of which has been invited to a technological symposium organized by fellow on European Society of Biomaterials (ESB) 2022 conference held in Bordeaux.