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3D Scaffolds as a Stem Cell Delivery System for Musculoskeletal Regenerative Medicine

Periodic Reporting for period 4 - CELL HYBRIDGE (3D Scaffolds as a Stem Cell Delivery System for Musculoskeletal Regenerative Medicine)

Reporting period: 2019-11-01 to 2020-04-30

Aging worldwide population demands new solutions to permanently restore damaged tissues, thus reducing healthcare costs. Regenerative medicine offers alternative therapies for tissue repair. Although first clinical trials revealed excellent initial response after implantation of these engineered tissues, long-term follow-ups demonstrated that degeneration and lack of integration with the surrounding tissues occur. Causes are related to insufficient cell-material interactions and loss of cell potency when cultured in two-dimensional substrates, among others.
Stem cells are a promising alternative due to their differentiation potential into multiple lineages. Yet, better control over cell-material interactions is necessary to maintain tissue engineered constructs in time. It is crucial to control stem cell quiescence, proliferation and differentiation in three-dimensional scaffolds while maintaining cells viable in situ. Stem cell activity is controlled by a complex cascade of signals called “niche”, where the extra-cellular matrix (ECM) surrounding the cells play a major role. Designing scaffolds inspired by this cellular niche and its ECM may lead to engineered tissues with instructive properties characterized by enhanced homeostasis, stability and integration with the surrounding milieu.
This research proposal aims at engineering constructs where scaffolds work as stem cell delivery systems actively controlling cell quiescence, proliferation, and differentiation. This challenge will be approached through a biomimetic design inspired by the mesenchymal stem cell niche. Three different scaffolds will be combined to achieve this purpose: (i) a scaffold designed to maintain cell quiescence; (ii) a scaffold designed to promote cell proliferation; and (iii) a scaffold designed to control cell differentiation. To prove the design criteria, the evaluation of stem cell quiescence, proliferation, and differentiation will be assessed for musculoskeletal regenerative therapies.
The project has been completed in all its objectives. We successfully developed a tri-compartment construct with the potential to regenerate osteochondral tissue.
For the first compartment of CELL HYBRIDGE, we developed hydrogels with different biological moieties that were screened to maintain cell quiescence. Cell quiescence was achieved, dependent on the chemistry composition of the hydrogels. The biological mechanisms behind the observed cell quiescence was elucidated and correlated to hydrogel physico-chemical properties.
For the second compartment of CELL HYBRIDGE, we developed different methods to control the fiber size, shape, and surface properties, to influence cell proliferation. We found a way to vary these scaffolds parameters spanning from obtaining homogeneous scaffolds to gradient scaffolds. We found that biological functionalization is not always needed to enhance cell proliferation. We correlated our findings to the biological mechanisms behind enhanced (or maintained) cell proliferation. We developed a method to increase cell migration capacity throughout the scaffolds, achieving full cell migration over 2mm in thickness in vivo, which is unreported so far with nanofibrillar meshes.
We found that cells in 3D express much lower levels of lamins compared to conventional 2D cultures, which we correlated to a specific focal adhesion protein. Such differential expression of lamins seems also to be connected to a differential migratory potential of cells.
For the third compartment of CELL HYBRIDGE, we developed protocols to bind biological moieties to 3D printed scaffolds and create inverse gradients of osteogenic and chondrogenic peptides. We also conducted proteomics studies to discover the protein signature on 3D scaffolds for osteochondral differentiation. Interestingly, we seemed to have found an indication that the protein signature in osteochondral cell differentiation is independent from the biomaterial chemistry analyzed, which may shed light on a broader applicability to different biomaterial scaffolds of the methodology that we have here developed.
We successfully integrated the 3 different compartments, obtaining the final CELL HYBRIDGE construct. In vitro studies showed that it was possible to obtain selective osteogenic and chondrogenic cell differentiation moving from the bony side to the cartilage side of the osteochondral scaffolds. In vivo studies further confirmed the in vitro studies both in small animals in an ectopic location as well as in large animals in an orthotopic skeletal location. Furthermore, small animal studies also allowed us to further confirm the biocompatibility of all compartments, used alone or in the integrated CELL HYBRIDGE construct.

Results were disseminated at several international and national conferences over the 5 years of duration of the project, extending the ERC results not only to European events, but also to the other continents. 3 PhD. thesis will be completed within the course of the next months. Originating from the work of the team, 12 scientific papers have already been published in international peer reviewed journals, and approximately 10-12 more are expected.
"The development of a hydrogel able to maintain adult stem cell in a quiescent state is probably the most significant finding we made in the past 5 years.
The use of 3D scaffolds as a more fundamental tool for studying biology in 3D has also been an important byproduct of the grant, showing that there is still so much unknown on cell behavior in 3D compared to conventional 2D culture systems. This also led to the unconventional findings that well known proliferative growth factors, such as FGF-1, may not be needed in the 3D scaffolds used in CELL HYBRIDGE. Despite we cannot generalize for all 3D scaffolds and 3D cell culture systems currently available, our findings certainly provide an important indication for others to also further investigate in this direction.
The integration of the hydrogel compartment with the nanofibrillar mesh compartment, which formed almost a cup around the hydrogel, provided the unexpected exciting result that it was possible to maintain stem cell viability for much longer time in vivo than what currently possible through simpler forms of delivery (e.g. injection). This could open new avenues to establish more effective cell therapy strategies in different applications where the use of the secretome of stem cells as a ""cell factory"" is of interest.
Finally, the full construct of CELL HYBRIDGE showed promising results in terms of osteochondral regeneration. While several studies have shown the development of interesting scaffold-based strategies for osteochondral regeneration, their translation to the clinic remain elusive due to lack of proper control over the mechanical and biological properties of the fabricated scaffolds, consequently resulting in a lack of control of cell activity. Despite we are not yet able to confirm if our constructs will maintain the promise in clinical trials, the fabricated scaffolds showed to support the right conducive biomechanical environment to control cell activity. In vivo results showed osteochondral tissue regeneration and integration with surrounding tissues, which is a promising step towards more durable regenerative medicine solutions."
Overall concept of CELL HYBRIDGE scaffolds for skeletal regeneration