Liquefied capsules containing microparticles with variable stiffness to control cell differentiation

• Autor(a) principal: Pinheiro, Diogo Marques
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/30916
• topic: Adipose tissue; Articular cartilage; Bioencapsulation; Bone; Cell differentiation; Mechanotransduction; Mesenchymal stem/stromal cells; Microparticles; Stiffness; Tissue engineering

Tissue engineering and regenerative medicine (TERM) strategies make use of selective cell differentiation to engineer biomimetic tissues through a series of factors, with a recent focus on mechanotransduction-based differentiation. Such mechanobiology-based TERM (or Mech-TERM) strategies make use of the mechanical forces in any given system to steer tissue formation towards desired pathways. This is performed by attempting to emulate the forces present in the tissue that we want to replicate, bringing as such an entirely new layer of biomimicry to TE systems. One such form of systems is bioencapsulation with liquefied capsules, due to its highly customizable nature and potential for application through minimally invasive procedures. This type of system also ensures high cell viability and proliferation, as well as immunoprotection. In this work, human adipose stem cells (hASCs) were co-encapsulated with microparticles of distinct stiffnesses, acting as 3D scaffolds for cellular adhesion and development. Our hypothesis is that scaffolds with stiffnesses emulating those of bone or articular cartilage will be able to guide cell differentiation into osteogenic or chondrogenic lineages, respectively, through the process of mechanotransduction. For that, hASCs were co-encapsulated with gelatin-grafted polycaprolactone (PCL) microparticles (STIFF+ capsules) or with gelatin microparticles (STIFF- capsules) and cultured for 28 days in basal (BAS), osteogenic (OST), and chondrogenic (CHO) differentiation media, followed by a series of analysis to determine the extent of cell viability, proliferation, and differentiation into the osteogenic and chondrogenic lineages. This system showed that high cell viability and proliferation were overall maintained, and that traces of osteogenic and chondrogenic differentiation were observed in both capsule types. Hydroxyapatite (HA) formation was detected in STIFF+ capsules cultured in BAS and OST media, and in STIFF- capsules in OST medium, at 21 days. Collagen type II was detected in both capsule types and in all media, in different amounts, at day 28. Alkaline phosphatase (ALP) activity and sulfated glycosaminoglycans (sGAG) content profiles overall indicated that STIFF+ and STIFF- capsules had, respectively, favored osteogenic and chondrogenic differentiation. Future studies involving more specific assays, such as identifying and/or quantifying expression of key osteogenic and chondrogenic genes and pathways in hASCs, are suggested to fully validate this hypothesis.