Alain da Silva Morais

Tumor Growth Suppression Induced by Biomimetic Silk Fibroin Hydrogels.

Protein-based hydrogels with distinct conformations which enable encapsulation or differentiation of cells are of great interest in 3D cancer research models. Conformational changes may cause macroscopic shifts in the hydrogels, allowing for its use as biosensors and drug carriers. In depth knowledge on how 3D conformational changes in proteins may affect cell fate and tumor formation is required. Thus, this study reports an enzymatically crosslinked silk fibroin (SF) hydrogel system that can undergo intrinsic conformation changes from random coil to β-sheet conformation. In random coil status, the SF hydrogels are transparent, elastic, and present ionic strength and pH stimuli-responses. The random coil hydrogels become β-sheet conformation after 10 days in vitro incubation and 14 days in vivo subcutaneous implantation in rat. When encapsulated with ATDC-5 cells, the random coil SF hydrogel promotes cell survival up to 7 days, whereas the subsequent β-sheet transition induces cell apoptosis in vitro. HeLa cells are further incorporated in SF hydrogels and the constructs are investigated in vitro and in an in vivo chick chorioallantoic membrane model for tumor formation. In vivo, Angiogenesis and tumor formation are suppressed in SF hydrogels. Therefore, these hydrogels provide new insights for cancer research and uses of biomaterials.

Silk-based anisotropical 3D biotextiles for bone regeneration

Bone loss in the craniofacial complex can been treated using several conventional therapeutic strategies that face many obstacles and limitations. In this work, novel three-dimensional (3D) biotextile architectures were developed as a possible strategy for flat bone regeneration applications. As a fully automated processing route, this strategy as potential to be easily industrialized. Silk fibroin (SF) yarns were processed into weft-knitted fabrics spaced by a monofilament of polyethylene terephthalate (PET). A comparative study with a similar 3D structure made entirely of PET was established. Highly porous scaffolds with homogeneous pore distribution were observed using micro-computed tomography analysis. The wet state dynamic mechanical analysis revealed a storage modulus In the frequency range tested, the storage modulus values obtained for SF-PET scaffolds were higher than for the PET scaffolds. Human adipose-derived stem cells (hASCs) cultured on the SF-PET spacer structures showed the typical pattern for ALP activity under osteogenic culture conditions. Osteogenic differentiation of hASCs on SF-PET and PET constructs was also observed by extracellular matrix mineralization and expression of osteogenic-related markers (osteocalcin, osteopontin and collagen type I) after 28 days of osteogenic culture, in comparison to the control basal medium. The quantification of convergent macroscopic blood vessels toward the scaffolds by a chick chorioallantoic membrane assay, showed higher angiogenic response induced by the SF-PET textile scaffolds than PET structures and gelatin sponge controls. Subcutaneous implantation in CD-1 mice revealed tissue ingrowths accompanied by blood vessels infiltration in both spacer constructs. The structural adaptability of textile structures combined to the structural similarities of the 3D knitted spacer fabrics to craniofacial bone tissue and achieved biological performance, make these scaffolds a possible solution for tissue engineering approaches in this area.

Combinatory approach for developing silk fibroin scaffolds for cartilage regeneration

Several processing technologies and engineering strategies have been combined to create scaffolds with superior performance for efficient tissue regeneration. Cartilage tissue is a good example of that, presenting limited self-healing capacity together with a high elasticity and load-bearing properties. In this work, novel porous silk fibroin (SF) scaffolds derived from horseradish peroxidase (HRP)-mediated crosslinking of highly concentrated aqueous SF solution (16 wt%) in combination with salt-leaching and freeze-drying methodologies were developed for articular cartilage tissue engineering (TE) applications. The HRP-crosslinked SF scaffolds presented high porosity (89.3 ± 0.6%), wide pore distribution and high interconnectivity (95.9 ± 0.8%). Moreover, a large swelling capacity and favorable degradation rate were observed up to 30 days, maintaining the porous-like structure and β-sheet conformational integrity obtained with salt-leaching and freeze-drying processing. The in vitro studies supported human adipose-derived stem cells (hASCs) adhesion, proliferation, and high glycosaminoglycans (GAGs) synthesis under chondrogenic culture conditions. Furthermore, the chondrogenic differentiation of hASCs was assessed by the expression of chondrogenic-related markers (collagen type II, Sox-9 and Aggrecan) and deposition of cartilage-specific extracellular matrix for up to 28 days. The cartilage engineered constructs also presented structural integrity as their mechanical properties were improved after chondrogenic culturing. Subcutaneous implantation of the scaffolds in CD-1 mice demonstrated no necrosis or calcification, and deeply tissue ingrowth. Collectively, the structural properties and biological performance of these porous HRP-crosslinked SF scaffolds make them promising candidates for cartilage regeneration. STATEMENT OF SIGNIFICANCE In cartilage tissue engineering (TE), several processing technologies have been combined to create scaffolds for efficient tissue repair. In our study, we propose novel silk fibroin (SF) scaffolds derived from enzymatically crosslinked SF hydrogels processed by salt-leaching and freeze-drying technologies, for articular cartilage applications. Though these scaffolds, we were able to combine the elastic properties of hydrogel-based systems, with the stability, resilience and controlled porosity of scaffolds processed via salt-leaching and freeze-drying technologies. SF protein has been extensively explored for TE applications, as a result of its mechanical strength, elasticity, biocompatibility, and biodegradability. Thus, the structural, mechanical and biological performance of the proposed scaffolds potentiates their use as three-dimensional matrices for cartilage regeneration.

Clinical Trials and Management of Osteochondral Lesions

Osteochondral lesions are frequent and important causes of pain and disability. These lesions are induced by traumatic injuries or by diseases that affect both the cartilage surface and the subchondral bone. Due to the limited cartilage ability to regenerate and self-repair, these lesions tend to gradually worsen and progress towards osteoarthritis. The clinical, social, and economic impact of the osteochondral lesions is impressive and although therapeutic alternatives are under discussion, a consensus is not yet been achieved. Over the previous decade, new strategies based on innovative tissue engineering approaches have been developed with promising results. However, in order those products reach the market and help the actual patient in an effective manner, there is still a lot of work to be done. The current state of the implications, clinical aspects, and available treatments for this pathology, as well as the ongoing preclinical and clinical trials are presented in this chapter.

Cartilage Tissue Engineering and Regenerative Strategies

Human adult articular cartilage is a unique avascular tissue which displays the ability to resist to repetitive compressive stress. However, this connective tissue exhibits slight capacity for intrinsic restoration and, then even injuries or lesions can lead to progressive damage and osteoarthritic joint deterioration. Therefore, the field of cartilage repair continues to expand, bridging the gap between palliative care and chondral defects reconstruction. Tissue engineering strategy, centered on three actors: cells, proteins and scaffolds, received a lot of attention in the aim to develop an articular cartilage regeneration process that will be efficient, simple, and based on global market, cost-effective. The current state of cartilage tissue engineering with respect to different cell-sources, growth factors and biomaterial scaffolds, as well as the strategies employed in the restoration and repair of damaged articular cartilage will be the focus of this book chapter.

Small Animal Models

Animal assays represent an important stage between in vitro studies and human clinical applications. These models are crucial for biomedical research and regenerative medicine studies, as these offer precious information for systematically assessing the efficacy and risks of recently created biomaterials, medical devices, drugs, and therapeutic modalities prior to initiation of human clinical trials. Therefore, selecting a suitable experimental model for tissue engineering purposes is essential to establish valid conclusions. However, it remains important to be conscious of the advantages and limitations of the various small and large animal models frequently used for biomedical research as well as the different challenges encountered in extrapolating data obtained from animal studies and the risks of misinterpretation. This chapter discusses the various small animal model strategies used for osteochondral defect repair. Particular emphasis will be placed on analyzing the materials and strategies used in each model.

In vitro and in vivo performance of methacrylated gellan gum hydrogel formulations for cartilage repair*: IN VITRO AND IN VIVO PERFORMANCE OF METHACRYLATED GG HYDROGEL FORMULATIONS

Methacrylated gellan gum (GGMA) formulation is proposed as a second-generation hydrogel for controlled delivery of cartilage-forming cells into focal chondral lesions, allowing immediate in situ retention of cells and 3D filling of lesion volume, such approach deemed compatible with an arthroscopic procedure. Formulation optimization was carried out in vitro using chondrocytes and adipose mesenchymal stromal/stem cells (ASCs). A proof-of-concept in vivo study was conducted using a rabbit model with induced chondral lesions. Outcomes were compared with microfracture or non-treated control. Three grading scores were used to evaluate tissue repair after 8 weeks by macroscopic, histological and immunohistochemical analysis. Intense collagen type II and low collagen type I gene and protein expression were achieved in vitro by the ASC + GGMA formulation, in light with development of healthy chondral tissue. In vivo, this formulation promoted significantly superior de novo cartilage formation compared with the non-treated group. Maintenance of chondral height and integration with native tissue was further accomplished. The physicochemical properties of the proposed GGMA hydrogel exhibited highly favorable characteristics and biological performance both in vitro and in vivo, positioning itself as an attractive xeno-free biomaterial to be used with chondrogenic cells for a cost-effective treatment of focal chondral lesions.

Tunable Enzymatically Cross-Linked Silk Fibroin Tubular Conduits for Guided Tissue Regeneration

Hollow tubular conduits (TCs) with tunable architecture and biological properties are in great need for modulating cell functions and drug delivery in guided tissue regeneration. Here, a new methodology to produce enzymatically cross-linked silk fibroin TCs is described, which takes advantage of the tyrosine groups present in silk structure that are known to allow the formation of a covalently cross-linked hydrogel. Three different processing methods are used as a final step to modulate the properties of the silk-based TCs. This approach allows to virtually adjust any characteristic of the final TCs. The final microstructure ranges from a nonporous to a highly porous network, allowing the TCs to be selectively porous to 4 kDa molecules, but not to human skin fibroblasts. Mechanical properties are dependent both on the processing method and thickness of the TCs. Bioactivity is observed after 30 days of immersion in simulated body fluid only for the TCs submitted to a drying processing method (50 °C). The in vivo study performed in mice demonstrates the good biocompatibility of the TCs. The enzymatically cross-linked silk fibroin TCs are versatile and have adjustable characteristics that can be exploited in a variety of biomedical applications, particularly in guidance of peripheral nerve regeneration.

Cell culture methods

The restoration of osteochondral defects presents great challenges that have not been fully solved by the current therapies. Therefore, this field continues to expand, bridging the gap between palliative care and defects reconstruction. In the last few years, tissue engineering and regenerative medicine have been offering advanced strategies and some of which have successfully reached clinical application and the market. Beyond the origin and source of cells, the development of culture conditions remains an important step to further clinical applications. Several approaches have been focused on good manufacturing practice (GMP) conditions. The aim is the creation of advanced therapy medicinal products (ATMPs). The up-to-date state of the culture protocols for osteochondral tissue engineering with respect to different cells, growth factors, and biomaterial scaffolds, as well as the strategies employed in clinical trials for the restoration and repair of osteochondral defects, will be the focus of this book chapter.