Fish scales as model for osteogenic cells culture

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Biomateriais, Reabilitação e Biomecânica)Considering the fish scales composition of hydroxyapatite and type I collagen fibrils, the organized and regular pattern of the scales, and also their capability to provide a form of armor plating to protect fish from injury and disease transmission, its potential biomedical application might be striking. In this work, fish scales of the specie Lates Calcarifer, also known as White Seabass, were studied, in particular by the assessment of their potential as model for osteogenic cells culture. For this purpose the fish scales were collected in a market in Thailand. The scales characterization, especially concerning their pattern, was done by observations in SEM and Confocal Microscope. Results showed that the overall scales pattern is formed by parallel concentric lines and is highly composed of collagen. An in vitro biological analysis of the scales when used as films to support cell (osteoblasts) proliferation was also done. In order to infer about the scales patterning potential on the proliferation of cells, fibroin films with the same pattern of the fish scales were produced, as well as and films without any pattern. To assess cell viability and cell proliferation, a technique denominated Alamar Blue was performed, being the cell proliferation higher on the scales, comparing with the fibroin films. Cell adhesion morphology, cytoskeleton architecture and alignment were evaluated by observations in the SEM and Confocal Microscope. The osteoblasts presented an orientation according to the fish scales pattern alignment and acquired an elongated and narrow shape. The DNA quantification and ALP test allowed drawing conclusions about the cell activity. The cells on the fibroin samples did not show any ALP activity during the 14 days of proliferation, although the ones on the scales already showed some activity after 14 days of the cells seeding. An in vitro biological analysis of the fish scales using human adipose-derived stem cells (hASCs) was executed, in the presence or absence of osteogenic differentiation factors. The stem cells behavior was very similar to the osteoblasts, presenting a strait and elongated shape aligned with the pattern axis. Results suggest that the fish scales composition and topography are responsible for a good cell proliferation and induce modifications in cell organization and morphology.Tendo em conta a composição das escamas de peixe em hidroxiapatite e fibras de colagénio do tipo I, o padrão organizado e regular das escamas, e também a sua capacidade de proporcionar uma forma de blindagem para proteger o peixe de lesões e transmissão de doenças, a sua aplicação biomédica poderá ser espantosa. Neste trabalho foram estudadas escamas de peixe da espécie Lates Calcarifer, referente ao Robalo Asiático, em particular a avaliação do seu potencial como modelos para cultura de células osteogénicas. Para este efeito, as escamas de peixe foram trazidas de um mercado na Tailândia. A caracterização das escamas, tendo predominantemente em conta o seu padrão, foi feita através de observações no SEM e Microscópio Confocal. Os resultados mostraram que o padrão global das escamas é constituído por linhas concêntricas paralelas e altamente composto por colagénio. Uma análise biológica in vitro das escamas quando usadas como scaffolds para suporte de proliferação celular (osteoblastos) também foi realizada. A fim de inferir acerca do potencial das escamas na proliferação dessas células foram também produzidos filmes de fibroína com o mesmo padrão das escamas e filmes sem qualquer padrão. Para avaliar a viabilidade e proliferação celular foi realizada uma técnica denominada “Alamar Blue”, tendo sido a proliferação celular maior nas escamas comparando com os filmes de fibroína. A morfologia de adesão celular, arquitectura do citoesqueleto e alinhamento foram avaliados por observações no SEM e Microscópio Confocal. Os osteoblastos apresentaram uma orientação de acordo com o alinhamento do padrão das escamas e adquiriram uma forma alongada e estreita. A quantificação de DNA e teste de ALP permitiram tirar conclusões acerca da actividade celular. As células nas amostras de fibroína não mostraram qualquer actividade celular durante os 14 dias de proliferação, contudo nas escamas mostraram já alguma actividade 14 dias depois do início da cultura das células. Uma nova análise in vitro das escamas foi feita com células estaminais do tecido adiposo, na presença e ausência de factores de diferenciação osteogénica. O comportamento das células estaminais foi semelhante ao dos osteoblastos, apresentando uma forma estreita e alongada e alinhadas com o eixo do padrão. Os resultados sugerem que a composição e topografia das escamas são responsáveis por boa proliferação celular e por induzirem modificações na morfologia e organização celular

Cell sheet engineering for reproducing the bone marrow hematopoietic stem cell niche

Hematopoietic stem and progenitor cells (HSPC) are multipotent cells responsible for the maintenance and renewal of the hematopoietic lineage in the adult body. The fate of these stem cells is closely regulated by their surrounding microenvironment, or niche. The importance of the microenvironment for HSPC function has been long recognized by researchers that more than 30 years ago attempted to emulate it in 2D using a layer of bone marrow stromal cells to culture hematopoietic cells for long time periods (Dexter-type cultures). However, all the models based on feeder layers are less than perfect in recreating the hematopoietic microenvironment. The use of growth factor cocktails provided some promising results concerning the maintenance and proliferation of some cell populations but still struggle to deliver the correct microenvironment for the maintenance of suitable HSPC populations. Part of the problem of the current systems lies on the lack of the third dimension. At the same time, the proposed three-dimensional methodologies using scaffolds to engineer the bone marrow (BM) microenvironment present very limited results probably due to the scaffolding matrices’ intrinsic limitations. Therefore, an engineered BM microenvironment capable of acting as a functional HSPC niche would provide a tremendous tool for the study of hematopoiesis as well as for obtaining and maintaining HSPC. Using osteogenic cell sheets, we have previously demonstrated that it was possible to induce the ectopic formation of mature bone tissue with a clear bone marrow, avoiding the use of scaffolds. In the present work, we studied the potential of using osteogenic cell sheets to build in vitro, a 3D microenvironment capable of providing HSPC a suitable niche for their survival and proliferation. For this, we used bone marrow stromal cells and adipose-derived stem cells to produce the osteogenic cell sheets and human umbilical cord blood as a source of hematopoietic stem cells