Coordinatore
Leonardo Ricotti
Scientific coordinator
Leonardo Ricotti is Associate Professor of Bioengineering and Biorobotics at SSSA and head of the “Micro-nano-bio systems and targeted therapies” Lab at the BioRobotics Institute. He holds a M.Sc. course on “Miniaturized therapeutic and regenerative technologies” and a PhD course on “Micro-nano-bio systems for medical and technological applications”. He has supervised or co-supervised 10 PhD students, working on therapeutic micro-devices, biomaterials and artificial organs, and 30 M.Sc. theses on bioengineering topics. He carries out innovative research efforts at the interface between different disciplines, such as robotics and mechatronics, materials science, molecular biology and biotechnology and he aims at creating innovative (and potentially disruptive) “match points” between different disciplines. He is co-author of ~90 scientific publications (60 on ISI journals), 6 book chapters on micro-nano systems for biomedical applications. He is also inventor of 9 patents. He is Associate Editor of the IEEE Transactions on NanoBioscience and of the IEEE Transactions on Medical Robotics and Bionics. In 2012, he received the “Massimo Grattarola” award for the best PhD Thesis in bioengineering (Thesis title: “Development of bio-hybrid actuators”). In July 2014, he was awarded with the European Biomaterials and Tissue Engineering Doctoral Award. In 2018, he received regional and national prizes as member of the spin-off company Relief s.r.l. He currently coordinates an European project (ADMAIORA – ADvanced nanocomposite MAterIals fOr in situ treatment and ultRAsound-mediated management of osteoarthritis), funded in the H2020 framework.Link al CV
Lorenzo Vannozzi
Technical Project Manager
Lorenzo Vannozzi is a post-doctoral fellow at SSSA, within the “Micro-nano-bio systems and targeted therapies” Lab of the BioRobotics Institute. In 2013, he received a Master degree in Biomedical Engineering at University of Pisa, with a thesis entitled “Design and development of a 3D system for bio-hybrid actuation” and in 2017 he obtained a PhD in Biorobotics, defending a thesis entitled “Novel actuated microsystems”. His research activity deals with the exploration of 3D microfabrication technologies, included 3D bioprinting, for bioengineering purposes, and the design, development and testing of drug delivery platforms for local therapies. He has an interdisciplinary approach involving materials science, mechatronics and molecular biology. He supported the teaching activity of Prof. Leonardo Ricotti within the M.Sc course on “Miniaturized therapeutic and regenerative technologies”, with practical classes on material synthesis and characterization. He is author or co-author of 13 scientific publications. In 2018, he received the “Julia Polak European Doctorate Award” from the European Society of Biomaterials committee. He is or has been involved in different Italian and European projects (MOTU, M2Neural and GeT Small), for which he provided important technical contributions.Link al CV
OPTIMIZATION OF THE PEG – AND PLURONIC – BASED HYDROGELS
D3.1 PEG-fibrinogen based hydrogel prototype development
In this Deliverable the prototypes of PEG-fibrinogen-based hydrogel formulations developed by REGENTIS and their main features are described. In section 2, the key specifications identified for the hydrogels are reported, also with a description of the three material types developed, as well as of the methods and techniques used to optimize the hydrogel formulations based on the required specifications. Then, in section 2.1, the results of preliminary biological tests performed to assess the viability of human adipose tissue-derived stem cells (ASCs) after cell encapsulation within PEGfibrinogen printed hydrogels are reported. These tests also served to evaluate the right amount of photoinitiator, UV intensity and exposure time to be employed to have a material respecting the specifications. After this preliminary analysis, an optimization process for PEG-based hydrogel formulations is described in the same section. This implied to vary: i) the total amount of Pluronic and ii) the ratio of non-cross-linkable and UVA-cross-linkable Pluronic. Material characterization in terms of mechanical and rheological properties, the diffusivity of proteins in the material and cell viability of human chondrocytes are also reported in section 2.1 for the different formulations. Results indicate that the formulations containing a lower amount of crosslinkable Pluronic show a higher swelling, a lower shear modulus (G') and a smaller Young modulus (E), corresponding to a few kPa. Results on protein diffusivity, obtained by testing some selected formulations, are reported in section 2.2 and suggest that the decreased amount of Pluronic increases the “permeability” of the material to nutrients available in the medium surrounding the hydrogels. However, even the formulation showing the highest diffusivity between the ones tested, does not guarantee a sufficient level of human chondrocytes cell viability, highlighting a possible relevant role played by material chemistry, with the tested cell type. In conclusion, the PEG-fibrinogen-based hydrogels do not result in good candidates usable as the matrix of the nanocomposite materials envisaged in ADMAIORA.
In this Deliverable the results obtained from the characterization of the PEG-fibrinogen based
hydrogels developed by REGENTIS are reported. In particular:
1. Material 1: A patented, bio-synthetic finely tuned combination of PEG-DA and
denatured human fibrinogen, crosslinkable through UV light, which is a liquid
formulation, also commercialized by REGENTIS with the name of Gelrin C.
2. Material 2: A patented, injectable and curable PEG-fibrinogen/Pluronic paste which is
based on PEG-fibrinogen conjugate, Pluronic F127 polymer and a photo-initiator.
Preliminary biological tests performed for assessing the viability of human adipose tissuederived stem cells (ASCs) highlighted a modification of cells morphology to spindle-like shape
in Material 1 (both UV-cured and non UV-cured), which resulted not printable, due to too low
viscosity. Thus, such material was excluded from further analyses.
Material 2 was printed with cells and different amounts of photoinitiator were tested. Results
from Live/Dead assay and MTT test confirmed a good viability and methabolic activity for an
Irgacure2959 concentration of 0.05%, compatible with a lower level of crosslinking, but that
was enough for retaining cells in the hydrogel. Subsequent experiments with ASCs cultured
in chondrogenic medium, however, highlighted a low viability after printing also in the
presence of TGFβ3, with almost all cells dead after 14 days of culture. This behaviour can be
attributed to chemical cues and also to a too high level of crosslinking of the material that led
to a small diffusion of nutrients, as demonstrated from the results obtained testing the
penetration of the BSA-FITC protein in some selected formulations of Material 2. REGENTIS
worked to modify the material formulation in order to match the requirements imposed by
the project, by varying: i) the total amount of Pluronic and ii) the ratio of non-cross-linkable
and UVA-cross-linkable Pluronic (i.e. Pluronic-OH and Pluronic-diacrylate, respectively).
Rheological tests and mechanical characterization results indicated that the formulations
containing a lower amount of cross-linkable Pluronic had a more significant swelling and a
lower shear modulus (G') and a smaller Young modulus (E), corresponding to a few kPa.
Protein diffusivity was evaluated on a few selected formulations of Material 2. Results
highlighted an increased diffusivity of BSA-FITC protein correlated to a lower amount of
Pluronic. However, even the formulation that showed the highest protein diffusivity did not
guarantee a sufficient level of cell viability, as resulted from tests performed with human
chondrocytes. This suggested that the chemico-physical properties of this material are not
suitable for chondrocyte encapsulation.
From the results obtained, the hydrogels made from Material 1 and Material 2 did not result
in suitable candidates for the project purposes.
D3.2 Pluronic-fibrinogen based hydrogel prototype development
In this Deliverable, the main features of the Pluronic-fibrinogen based formulations
developed by REGENTIS are described.
In particular, in the Introduction of section 2 the main specifications identified for the
hydrogels are reported, also with a description of the three types of materials developed
and the methods and techniques used to optimize the basal hydrogel formulations, based
on the specifications. In section 2.1, the Pluronic-fibrinogen based hydrogel formulation
optimization is described, together with material characterization results, in terms of
mechanical and rheological properties. Diffusivity of proteins in the material and cell viability
on human chondrocytes are reported in section 2.2.
The material tended to shrink when incubated at 37°C in a saline solution after being crosslinked with UV light. No marked gradients of fluorescent proteins were observed, indicating
a good diffusivity of nutrients into the material. However, such a material formulation did
not guarantee a high level of cell viability, probably due to material chemistry. For this
reason, the Pluronic-fibrinogen based hydrogel did not result in a good candidate for the
project purposes.
In this Deliverable the results obtained from the characterization of the Pluronic-fibrinogen
based hydrogels developed by REGENTIS are reported. In particular, they concern Material
3, a patented thermosensitive Pluronic-F127-fibrinogen hydrogel, suitable for printing, also
known with the commercial name of GelrinV.
As in the case of PEG-fibrinogen based hydrogels described in the Deliverable 3.1, the
Pluronic-F127 based hydrogels formulations were tuned by changing the amount of Pluronic
F127-OH and Pluronic F127-di-acrylate leading to a change in the thermo-responsive
properties of each formulation.
From the two prepared batches, namely FF0038 and FF0039, rheological and mechanical
properties have been characterized in function of the UV curing time and temperature,
respectively. An inconsistence in the batch manufacturing emerged from the obtained
results. Also, batch FF0038 resulted more effective in being crosslinked with UV light
radiation in a time compatible with the ADMAIORA project requirements. For this reason,
the subsequent characterization has been performed only on that batch of material.
From swelling analysis resulted that Material 3 tends to shrink after 24 h at 37°C in a saline
solution, with a volume variation of -38%.
Protein diffusivity tests highlighted the absence of marked gradients. The BSA-FITC fully
permeated in the gel, without accumulating at the borders, as obtained in the case of
Material 2 (see Deliverable D3.1). However, even if this result was promising in view of cell
viability, Live/Dead assay performed on human chondrocytes encapsulated in Pluronicfibrinogen based hydrogels, showed a high number of dead cells. Thus, the Pluronicfibrinogen based hydrogels do not represent good candidates for the project purposes.