Final Activity Report Summary - OSTEOCHONDRAL TE (Tissue engineering of autologous ostechondral implants)
In this Marie-Curie action, we have designed a catalog of technologies for engineering structural tissues. These technologies can work cooperatively between themselves and together with a population of adult stem cells of great potential in tissue engineering, namely mesenchymal stem cells.
The catalog of technologies developed is the following:
(1) A new biomaterial based on mixtures of silk fibroin and hyaluronan. This new biomaterial combines many of the main advantages of these two biomacromolecules. It is strong, slowly biodegradable, and able to support cell and tissue growth. It is able to induce regenerative responses through interactions with cells, like hyaluronan does.
(2) Moreover, we have found that the blended composition presents some unique properties that arise from interactions between the pure materials. First, hyaluronan can cooperate with other environmental factors to induce or enhance a change in the molecular conformation of silk fibroin towards a stable beta-sheet form. The increase in content of silk fibroin molecules into a very stable conformation, together with the formation of some separated domains of pure hyaluronan, led to biopolymer structures with outstanding mechanical properties. This is a very desirable feature when designing scaffolds for load-bearing structures, such as most structural tissues.
We also observed that in contrast to pure silk fibroin or hyaluronan, scaffolds from these blends can be prepared simply by a technique consisting of freeze-drying a solution of the two biopolymers and treating the resulting solid matrix with methanol. In contrast to silk fibroin and hyaluronan that need porogens and processes to destroy them in order to create a porous microstructure suitable for tissue ingrowth, these blends could form it just by the above-mentioned technique. This seems to be the result from silk fibroin supercontraction in the presence of hyaluronan and methanol, and to domains of pure hyaluronan that collapse into thin films after methanol incubation and drying.
(3) We have also developed an electrospinning protocol that can be applied on bio-materials based on silk fibroin and lead to scaffold surfaces with nanoscale topography (300-800 nm) wide fibres. Modifying this electrospinning technique, we can align these fibres along one longitudinal axis. We have found out that this nanotopography has a profound impact on MSCs seeded on these surfaces. MSCs in nanostructured surfaces attach quicker and to a greater extent than to flat surfaces. Moreover, we are able to align these cells by aligning the fibres on the surface to which cells are attached. This alignment is not only observed for cell morphology but also in its cytoskeleton. Research is continuing in our group through collaboration with the Functinal Genomic Center of ETH Zurich to investigate the possible effect of such surfaces in the gene expression pattern of MSCs. This technology could be particularly promising for tissues where cell guiding or alignment is important: neuron reconnection, ligaments, muscles, etc.
4) We have also investigated the effect of some combinations of soluble factors on the differentiation of MSCs into lineages that are relevant for the regeneration of structural tissues. We identified that a combination of the drug dexamethasone with two cytokines allowed us to shift MSC differentiation pattern to fibrocytes (and ligament like tissue formation) or chondrocytes (and cartilage like tissue formation). These cytokines are transforming growth factor-beta1 (TGF) and insulin like growth factor-I (IGF). Moreover, we have found out that the IGF dosing regimen, i.e. the pattern of concentrations over the time used, can shift the MSC differentiation pattern.
The catalog of technologies developed is the following:
(1) A new biomaterial based on mixtures of silk fibroin and hyaluronan. This new biomaterial combines many of the main advantages of these two biomacromolecules. It is strong, slowly biodegradable, and able to support cell and tissue growth. It is able to induce regenerative responses through interactions with cells, like hyaluronan does.
(2) Moreover, we have found that the blended composition presents some unique properties that arise from interactions between the pure materials. First, hyaluronan can cooperate with other environmental factors to induce or enhance a change in the molecular conformation of silk fibroin towards a stable beta-sheet form. The increase in content of silk fibroin molecules into a very stable conformation, together with the formation of some separated domains of pure hyaluronan, led to biopolymer structures with outstanding mechanical properties. This is a very desirable feature when designing scaffolds for load-bearing structures, such as most structural tissues.
We also observed that in contrast to pure silk fibroin or hyaluronan, scaffolds from these blends can be prepared simply by a technique consisting of freeze-drying a solution of the two biopolymers and treating the resulting solid matrix with methanol. In contrast to silk fibroin and hyaluronan that need porogens and processes to destroy them in order to create a porous microstructure suitable for tissue ingrowth, these blends could form it just by the above-mentioned technique. This seems to be the result from silk fibroin supercontraction in the presence of hyaluronan and methanol, and to domains of pure hyaluronan that collapse into thin films after methanol incubation and drying.
(3) We have also developed an electrospinning protocol that can be applied on bio-materials based on silk fibroin and lead to scaffold surfaces with nanoscale topography (300-800 nm) wide fibres. Modifying this electrospinning technique, we can align these fibres along one longitudinal axis. We have found out that this nanotopography has a profound impact on MSCs seeded on these surfaces. MSCs in nanostructured surfaces attach quicker and to a greater extent than to flat surfaces. Moreover, we are able to align these cells by aligning the fibres on the surface to which cells are attached. This alignment is not only observed for cell morphology but also in its cytoskeleton. Research is continuing in our group through collaboration with the Functinal Genomic Center of ETH Zurich to investigate the possible effect of such surfaces in the gene expression pattern of MSCs. This technology could be particularly promising for tissues where cell guiding or alignment is important: neuron reconnection, ligaments, muscles, etc.
4) We have also investigated the effect of some combinations of soluble factors on the differentiation of MSCs into lineages that are relevant for the regeneration of structural tissues. We identified that a combination of the drug dexamethasone with two cytokines allowed us to shift MSC differentiation pattern to fibrocytes (and ligament like tissue formation) or chondrocytes (and cartilage like tissue formation). These cytokines are transforming growth factor-beta1 (TGF) and insulin like growth factor-I (IGF). Moreover, we have found out that the IGF dosing regimen, i.e. the pattern of concentrations over the time used, can shift the MSC differentiation pattern.