A. L. Oliveira

Macro/microporous silk fibroin scaffolds with potential for articular cartilage and meniscus tissue engineering applications.

This study describes the developmental physicochemical properties of silk fibroin scaffolds derived from high-concentration aqueous silk fibroin solutions. The silk fibroin scaffolds were prepared with different initial concentrations (8, 10, 12 and 16%, in wt.%) and obtained by combining the salt-leaching and freeze-drying methodologies. The results indicated that the antiparallel β-pleated sheet (silk-II) conformation was present in the silk fibroin scaffolds. All the scaffolds possessed a macro/microporous structure. Homogeneous porosity distribution was achieved in all the groups of samples. As the silk fibroin concentration increased from 8 to 16%, the mean porosity decreased from 90.8±0.9 to 79.8±0.3% and the mean interconnectivity decreased from 97.4±0.5 to 92.3±1.3%. The mechanical properties of the scaffolds exhibited concentration dependence. The dry state compressive modulus increased from 0.81±0.29 to 15.14±1.70 MPa and the wet state dynamic storage modulus increased by around 20- to 30-fold at each testing frequency when the silk fibroin concentration increased from 8 to 16%. The water uptake ratio decreased with increasing silk fibroin concentration. The scaffolds present favorable stability as their structure integrity, morphology and mechanical properties were maintained after in vitro degradation for 30 days. Based on these results, the scaffolds developed in this study are proposed to be suitable for use in meniscus and cartilage tissue-engineered scaffolding.

Sodium silicate gel as a precursor for the in vitro nucleation and growth of a bone-like apatite coating in compact and porous polymeric structures.

In the present work, a new methodology to produce bioactive coatings on the surface of starch-based biodegradable polymers or other polymeric biomaterials is proposed. A sodium silicate gel is employed as an alternative nucleating agent to the more typical bioactive glasses for inducing the formation of a calcium-phosphate (Ca-P) layer. The method has the advantage of being able to coat efficiently both compact materials and porous 3D architectures aimed at being used on tissue replacement applications and as tissue engineering scaffolds. By means of this treatment, it is possible to observe the formation of an apatite-like layer, only after 6 hours of simulated body fluid immersion. For the porous materials, this layer could also be observed inside the pores, clearly covering the cell walls. Furthermore, an increase of the surface hydrophilicity (higher amount of polar groups in the surface) might contribute to the formation of silanol groups that also act as apatite inductors. After 30 days of SBF immersion, the apatite-like films exhibit a partially amorphous nature and the Ca/P ratios became much closer to the value attributed to hydroxyapatite (1.67). The obtained results are very promising for the development of cancellous bone replacement materials and for pre-calcifying bone tissue engineering scaffolds.

Bilayered silk/silk-nanoCaP scaffolds for osteochondral tissue engineering : in vitro and In vivo assessment of biological performance

Novel porous bilayered scaffolds, fully integrating a silk fibroin (SF) layer and a silk-nano calcium phosphate (silk-nanoCaP) layer for osteochondral defect (OCD) regeneration, were developed. Homogeneous porosity distribution was achieved in the scaffolds, with calcium phosphate phase only retained in the silk-nanoCaP layer. The scaffold presented compressive moduli of 0.4MPa in the wet state. Rabbit bone marrow mesenchymal stromal cells (RBMSCs) were cultured on the scaffolds, and good adhesion and proliferation were observed. The silk-nanoCaP layer showed a higher alkaline phosphatase level than the silk layer in osteogenic conditions. Subcutaneous implantation in rabbits demonstrated weak inflammation. In a rabbit knee critical size OCD model, the scaffolds firmly integrated into the host tissue. Histological and immunohistochemical analysis showed that collagen II positive cartilage and glycosaminoglycan regeneration presented in the silk layer, and de novo bone ingrowths and vessel formation were observed in the silk-nanoCaP layer. These bilayered scaffolds can therefore be promising candidates for OCD regeneration.

Surface modification tailors the characteristics of biomimetic coatings nucleated on starch-based polymers.

This work describes the influence of surface pretreatments over the nucleation and growth of an apatite layer, formed by a biomimetic process, on which a bioactive glass is used as a precursor of the calcium-phosphate (Ca-P) formation on the materials surface. SEVA-C, a corn starch-based biodegradable blend, was used as substrate. The surfaces were pretreated during various periods by: (i) physical methods, namely ultraviolet radiation (u.v.), and over exposure to ethylene oxide sterilization (EtO); and (ii) chemical methods, namely potassium hydroxide (KOH) and acetic anhydride (CH3CO)2 etchings. The surface modifications, performed before the production of the biomimetic coatings, resulted in a faster formation of Ca-P nuclei during the first stages of SBF immersion, particularly in the case of the KOH etching. In this case, it was possible to observe a decrease in the average surface roughness, as measured by laser profilometry, and an increase of the hydrophilicity of the material, which was evident from a clear increment in the water-uptake ability and quantified by contact angle measurements. With this treatment it was possible not only to reduce the induction period for the formation of a well defined and dense apatite-like layer, as observed by scanning electron microscopy (SEM), but also to improve the adhesion of the Ca-P layer to the substrate, as confirmed by the adhesion strength tests. For all the studied pre-treatments, the composition of the films, analyzed by energy dispersive spectroscopy (EDS) and identified by thin-film X-ray diffraction (TF-XRD), seems to be very similar to that of human bone apatites.

Aligned silk-based 3-D architectures for contact guidance in tissue engineering.

An important challenge in the biomaterials field is to mimic the structure of functional tissues via cell and extracellular matrix (ECM) alignment and anisotropy. Toward this goal, silk-based scaffolds resembling bone lamellar structure were developed using a freeze-drying technique. The structure could be controlled directly by solute concentration and freezing parameters, resulting in lamellar scaffolds with regular morphology. Different post-treatments, such as methanol, water annealing and steam sterilization, were investigated to induce water stability. The resulting structures exhibited significant differences in terms of morphological integrity, structure and mechanical properties. The lamellar thicknesses were ∼2.6 μm for the methanol-treated scaffolds and ∼5.8 μm for water-annealed. These values are in the range of those reported for human lamellar bone. Human bone marrow-derived mesenchymal stem cells (hMSC) were seeded on these silk fibroin lamellar scaffolds and grown under osteogenic conditions to assess the effect of the microstructure on cell behavior. Collagen in the newly deposited ECM was found aligned along the lamellar architectures. In the case of methanol-treated lamellar structures, the hMSC were able to migrate into the interior of the scaffolds, producing a multilamellar hybrid construct. The present morphology constitutes a useful pattern onto which hMSC cells attach and proliferate for guided formation of a highly oriented extracellular matrix.

Bioactive macro/micro porous silk fibroin/Nano-sized calcium phosphate scaffolds with potential for bone tissue engineering applications

AIM The development of novel silk/nano-sized calcium phosphate (silk/nano-CaP) scaffolds with highly dispersed CaP nanoparticles in the silk fibroin (SF) matrix for bone tissue engineering. MATERIALS & METHODS Nano-CaP was incorporated in a concentrated aqueous SF solution (16 wt.%) by using an in situ synthesis method. The silk/nano-CaP scaffolds were then prepared through a combination of salt-leaching/lyophilization approaches. RESULTS The CaP particles presented good affinity to SF and their size was inferior to 200 nm when theoretical CaP/silk ratios were between 4 and 16 wt.%, as determined by scanning electron microscopy. The CaP particles displayed a uniform distribution in the scaffolds at both microscopic and macroscopic scales as observed by backscattered scanning electron microscopy and micro-computed tomography, respectively. The prepared scaffolds presented self-mineralization capability and no cytotoxicity confirmed by in vitro bioactivity tests and cell viability assays, respectively. CONCLUSION These results indicated that the produced silk/nano-CaP scaffolds could be suitable candidates for bone-tissue-engineering applications.

New biotextiles for tissue engineering: Development, characterization and in vitro cellular viability

This work proposes biodegradable textile-based structures for tissue engineering applications. We describe the use of two polymers, polybutylene succinate (PBS) proposed as a viable multifilamentand silk fibroin (SF), to produce fibre-based finely tuned porous architectures by weft knitting. PBS is here proposed as a viable extruded multifilament fibre to be processed by a textile-based technology. A comparative study was undertaken using a SF fibre with a similar linear density. The knitted constructs obtained are described in terms of their morphology, mechanical properties, swelling capability, degradation behaviour and cytotoxicity. The weft knitting technology used offers superior control over the scaffold design (e.g. size, shape, porosity and fibre alignment), manufacturing and reproducibility. The presented fibres allow the processing of a very reproducible intra-architectural scaffold geometry which is fully interconnected, thus providing a high surface area for cell attachment and tissue in-growth. The two types of polymer fibre allow the generation of constructs with distinct characteristics in terms of the surface physico-chemistry, mechanical performance and degradation capability, which has an impact on the resulting cell behaviour at the surface of the respective biotextiles. Preliminary cytotoxicity screening showed that both materials can support cell adhesion and proliferation. These results constitute a first validation of the two biotextiles as viable matrices for tissue engineering prior to the development of more complex systems. Given the processing efficacy and versatility of the knitting technology and the interesting structural and surface properties of the proposed polymer fibres it is foreseen that the developed systems could be attractive for the functional engineering of tissues such as skin, ligament, bone or cartilage.

Cell adhesion and proliferation on biomimetic calcium-phosphate coatings produced by a sodium silicate gel methodology

The present study describes a methodology to produce bioactive coatings on the surface of starch based biodegradable polymers or other polymeric biomaterials. As an alternative to the more typical bioactive glass percursors, a sodium silicate gel is being employed as a nucleating agent, for inducing the formation of a calcium-phosphate (Ca-P) layer. The method has the advantage of being able to coat efficiently both compact materials and porous 3D architectures aimed at being used on tissue replacement applications and as bone tissue engineering scaffolds. This treatment is also very effective in reducing the incubation periods, being possible to observe the formation of an apatite-like layer, only after 6 h of immersion in a simulated body fluid (SBF). The influence of the SBF concentration on the formation of the apatite coating was also studied. The apatite coatings formed under different conditions were analyzed and compared in terms of morphology, chemical composition and structure. After the first days of SBF immersion, the apatite-like films exhibit the typical cauliflower like morphology. With increasing immersion times, these films exhibited a partially amorphous nature and the Ca/P ratios became very closer to the value attributed to hydroxyapatite (1.67). The obtained results are very promising for pre-calcifying bone tissue engineering scaffolds. Therefore, in order to study cell behavior and response to these apatite coatings, adhesion, morphology, and proliferation of a human osteoblast cell line (SaOS-2) was also analyzed after being cultured in the coatings formed after 15 days of immersion in SBF. Results indicate a good correlation between crystallinity of the apatite like coatings formed in these conditions and respective cell spreading and morphology. In general, higher cell proliferation was observed for higher crystalline Ca-P coatings.

Peripheral mineralization of a 3D biodegradable tubular construct as a way to enhance guidance stabilization in spinal cord injury regeneration

Spinal cord injuries (SCI) present a major challenge to therapeutic development due to its complexity. Combinatorial approaches using biodegradable polymers that can simultaneously provide a tissue scaffold, a cell vehicle, and a reservoir for sustained drug delivery have shown very promising results. In our previous studies we have developed a novel hybrid system consisting of starch/poly-e-caprolactone (SPCL) semi-rigid tubular porous structure, based on a rapid prototyping technology, filled by a gellan gum hydrogel concentric core for the regeneration within spinal-cord injury sites. In the present work we intend to promote enhanced osteointegration on these systems by pre-mineralizing specifically the external surfaces of the SPCL tubular structures, though a biomimetic strategy, using a sodium silicate gel as nucleating agent. The idea is to create two different cell environments to promote axonal regeneration in the interior of the constructs while inducing osteogenic activity on its external surface. By using a Teflon cylinder to isolate the interior of the scaffold, it was possible to observe the formation of a bone-like poorly crystalline carbonated apatite layer continuously formed only in the external side of the tubular structure. This biomimetic layer was able to support the adhesion of Bone Marrow Mesenchymal Stem Cells, which have gone under cytoskeleton reorganization in the first hours of culture when compared to cells cultured on uncoated scaffolds. This strategy can be a useful route for locally stimulate bone tissue regeneration and facilitating early bone ingrowth.

De novo bone formation on macro/microporous silk and silk/nano-sized calcium phosphate scaffolds

Macro/microporous silk/nano-sized calcium phosphate scaffolds (SC16) with bioactive and superior physicochemical properties are currently being developed. In this study, we evaluated the new bone formation ability in rat femur of the SC16 scaffolds in vivo, using silk fibroin scaffolds (S16) as control. The CaP distribution profile in the scaffolds was characterized by micro-computed tomography and the CaP phase was found to be distributed homogeneously in the SC16 scaffolds. Mineralization was only observed in SC16 scaffolds, and both scaffolds gradually degraded with time. By staining the explants, new bone growth was observed directly on the SC16 surface and with higher density than that observed on the S16 scaffolds. These results demonstrated that the SC16 hybrid scaffolds are osteoconductive and can be good candidates for bone tissue engineering as they promote superior de novo bone formation.