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Printing Ultrasound Stimulated piezoelectric materials for Tissue Engineering

Periodic Reporting for period 1 - PRIUS-TE (Printing Ultrasound Stimulated piezoelectric materials for Tissue Engineering)

Reporting period: 2020-04-01 to 2022-03-31

On an aging society, our quality of life depends significantly in our capability to regenerate or engineer replacements for diseased and damaged tissues. One of these is the osteochondral interface. Over 30% of the population above the age of 65 is affected by osteochondral defects, being the most common cause of disability in older adults. PRIUS-TE (Printing Ultrasound Stimulated piezoelectric materials for Tissue Engineering) aims to regenerate the osteochondral interface with the use of hierarchical piezoelectric materials capable of stimulating mechanically, electrically and chemically the cells. Cartilage is unable to adequately self-regenerate due to its avascular character, the high content of extra cellular matrix (ECM) and the quiescent character of cells within (chondrocytes). Damage or diseases such as osteoarthritis (OA) lead to degeneration, reaching subchondral bone and generating an osteochondral defect. Clinical treatments rely on microfracture techniques that recruit tissue-specific progenitor (or stem) cells from the bone marrow, and form a de-novo cartilaginous tissue. However, the recruited cells are not able to self-organize and differentiate into phenotypically coherent cells. This results in the formation of unstructured and isotropic tissues with impaired mechanical properties that fail at long term. Current TE strategies are mainly based on isotropic materials that disregard the intrinsic multi-zonal character of the native tissue. PRIUS-TE takes inspiration from the structure and intrinsic properties of the osteochondral interface. It is based on hierarchical scaffolds that mimic the structure, cell microenvironment and fixed ionic charge responsible of the mechanical properties of the native tissue. These gradient, hierarchical and piezoelectric scaffolds will stimulate the recruited cells electrically, mechanically and chemically promoting the layer-specific cell growth, differentiation and the formation of a coherent tissue.
PRIUS-TE has developed hierarchical scaffolds with gradient piezoelectric and biochemical features with potential to regenerate the osteochondral interface through their activation with ultrasound stimulation.
The scaffolds are based on polylactide and polycaprolactone that phase segregate during the printing process. The phase segregation depended on the polymer ratio used for printing and the molecular weight of the polymers. Piezoelectricity was introduced by the inclusion of cellulose nanocrystals (CNCs) with high density of negative charges on the surface. For that, CNCs were isolated from filter paper and oxidize with TEMPO reactions that lead to carboxylated CNCs (cCNCs). Compatibilization of the nanoparticles with the polymeric matrix was attempted by grafting small PLA chains from the surface of the cCNCs (PLA-g-CNCs). However, atomic force microscopy and dynamic mechanical analysis demonstrated that bare cCNCs integrated better on the PLA matrix than PLA-g-CNCs. We hypothesized that this unexpected result was a consequence of the steric hindrance of the PLA short chains that disrupted the formation of ordered structures within the higher molecular weight PLA matrix. Bare cCNCs were mix with PLA and solvent casted to create homogeneous films that were later blended with PCL via extrusion. The blends were then used to feed a screw-driven printer that allowed the formation of CNC loaded PLA:PCL scaffolds. CNCs were loaded at different ratios on the polymer blend and the phase segregated structure remained unchanged at a microscopic level.
Inclusion of bioactive molecules on the shape of peptide sequences representative of chondrogenic environments (E-cadherin and collagen II) was done by end-group functionalization of the PCL with azide and maleimide moieties. After printing, these moieties were used to “click” the peptide sequences on the solid state. Control of the loaded PCL-maleimide or PCL-azide ratio and the PCL:PLA ratio allowed to control the amount of presented peptides on the surface of the scaffolds.
Stimulation of the scaffolds with ultrasounds could potentially activate piezoelectricity on the CNCs and initial cell cultures with human mesenchymal stem cells (hMSCs) show good biocompatibility. Activation of mechanotransduction markers such as Yess-associated protein was investigated showing no significant difference between groups.
Dissemination of the work was done at multiple scientific conferences (European Society for Biomaterials, 2021; 262nd annual meeting of the American Chemical Society and iCANX Talks) and through dedicated twitter account (@prius_te).
PRIUS-TE showed for the first time the possibility of creating hierarchical and piezoelectric scaffolds presenting biochemical gradients on 3D printing scaffolds. These scaffolds are potentially responsive to ultrasound stimulation, are biocompatible and could support the formation of a chondrogenic matrix.
The results could impact the society by providing an alternative solution to the treatment of osteochondral defects and by the development of new routes to tissue regeneration. These, if successful on in-vivo scenarios, could reduce the costs associated to recurrent surgeries due to long-term failure of current treatments, and the costs associated to reduced productivity of workers suffering from osteochondral defects.
Hierarchical piezoelectric scaffolds with Biochemical cues

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