Development of tissue engineered strategies combining stem cells and scaffolds aimed to regenerate bone and osteochondral interfaces

Abstract

Bone is a specialized tissue characterized by its rigidity and hardness, yet light weighed to fulfill diverse functions as mineral storage, organ protection or body support and locomotion. Despite its extraordinary healing ability, bone response may be unsuccessful to repair severe damage caused by injury or degenerative diseases. Furthermore, when bone is affected, other tissues and interfaces might be quite distressed as well. Cartilage and bone interface of the joints (osteochondral interfaces) is particularly affected by traumatic injuries and aging diseases. The challenge lies in balancing the structural, functional and biological needs of bone and cartilage in a stable milieu. As currently used therapies do not provide the ideal treatment, the development of biological substitutes through tissue engineering (TE) approaches may provide the ultimate solutions to restore, maintain, or improve bone and osteochondral tissue function. Scaffolds play an imperative role in most TE strategies, where they are expected to guide cellular distribution and colonization, similarly to the natural occurring communications between cells and tissue, and to provide mechanical support during tissue regeneration. Nevertheless, in large damaged areas, scaffolding alone might be insufficient to promote a satisfactory healing response. Culturing stem cells onto the scaffolds has demonstrated to promote the regeneration of damaged tissues. Stem cells (SCs) can be found in almost every tissue, evidencing their role in repairing injuries. Bone marrow stem cells (BMSCs) are the most studied, and promising candidates for autologous TE approaches minimizing disease transmission risks but shown to be donor age-affected and had limited self-renewal capability. These limitations directed research into other stem cells sources, such as amniotic fluid (AF), that have shown to be an almost unlimited SCs source with high proliferative and osteogenic potential. Along with the almost endless ability to expand without telomere shortening, amniotic fluid stem cells (AFSCs) share with embryonic stem cells some markers and a high self renewal capacity. In this Thesis several potential approaches were considered aiming at bone and osteochondral TE, focusing on distinct scaffold design and composition, previously or newly developed, and distinct stem cells sources. Different animal models were used to evaluate the proposed strategies with scaffolds and/or cell-scaffold constructs. As a first approach, a multilayered scaffold was developed composed of tricalcium phosphate (TCP) granules entrapped in a polycaprolactone (PCL) nanofiber mesh, inspired from the natural organic-inorganic nanostructure of bone. A synergistic effect of PCL-TCP scaffolds and mechanical stimulation was observed in the osteogenic differentiation of BMSCs cultured onto these scaffolds, resulting in the production of a mineralized ECM, even in basal medium. Composite multilayered scaffolds showed an interesting behavior under dynamic conditions using cell culturing media without osteogenic supplements. Another approach consisted in the combination of wet-spinning technology and a calcium silicate solution to produce SPCL (starch and polycaprolactone blend) wet-spun fiber meshes with functionalized silanol groups (SPCL-Si). The purpose was to developed new bioactive materials linking the properties of classical bioactive ceramics, and the processability and degradability of an organic polymer. SPCL-Si scaffolds own intrinsic properties to sustain in vitro osteogenic features, and thus holding a great potential for bone engineering approaches. Additionally, this Thesis aimed at designing a new construct for the repair of osteochondral (OC) interfaces, proposing a novel bilayered scaffold, combining the well described agarose gels for cartilage and the promising SPCL scaffolds for bone, encapsulated/seeded with amniotic stem cells (AFSCs). An OC engineered system was successfully developed, where both osteo- or chondrogenic differentiated AFSCs maintained long term viability and phenotypic expression, even in basal medium after assembling of the bilayered construct. Another major original objective of this Thesis was to explore the potential of amniotic fluid stem cells (AFSCs), as compared to bone marrow stem cells (BMSCs), for bone TE applications. Besides their source, the environmental conditions are known to influence cell response, and thus, both AFSCs and BMSCs were seeded/cultured in either 2D or 3D (using SPCL scaffolds) conditions. AFSCs and BMSCs expressed different bone-related markers at different time points. This study demonstrated that the selection of a particular stem cell type may not be a simple and direct process and relies on the target TE strategy. Finally, non-critical sized defects were induced in goat femurs so as to understand the role of the scaffold material -SPCL- and the influence of culturing autologous mesenchymal cells with/without pre-culture in osteogenic medium. Neobone formation and cellular distribution was increased in cell seeded SPCL scaffolds (pre-differentiation condition), showing the relevance of implanted cells in the bone regeneration process, and suggesting the importance of the stage of osteogenic differentiation of seeded cells. In a similar approach, femoral critical sized defects were induced in nude rats and SPCL scaffolds were implanted with or without AFSCs under different stages of osteogenic differentiation. The bridging effect between the bone segments was more prominent in scaffolds with osteogenic committed cells, and large blood vessels were observed, especially in SPCL scaffolds seeded with undifferentiated cells or osteogenic-like cells. Both in vivo studies showed the potential of SPCL scaffolds as a tissue 3D support for the regeneration of bone and underlined the importance of stem cells and stem cells stage of differentiation for achieving enhanced bone tissue regeneration. In summary, the described scaffold design and composition show a great potential to be tailored to specific applications in bone tissue regeneration strategies. Nevertheless, SPCL meshes obtained from melt spun fibers are clearly one step ahead as scaffold structures, showing to provide the necessary support for bone and osteochondral TE strategies using different sources of stem cells. Most importantly, the work described in this Thesis clearly demonstrated that bone tissue engineering requires the presence of stem cells, and that their pre-differentiation into the osteoblastic phenotype facilitates bone regeneration.