Bioencapsulation of stem and endothelial cells towards an in vitro model for endochondral ossification

• Autor(a) principal: Fernandes, Inês de Jesus
• 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/10773/31138
• topic: 3D co-culture; Bioencapsulation; Bone regeneration; Endochondral ossification; Endothelial cells; Dynamic culture; In vitro vascularization; Layer-by-layer; Mesenchymal stem cells; Tissue engineering; Umbilical cord

In the past, most bone tissue engineering (TE) strategies were focused on the recapitulation of intramembranous ossification (IMO) process. Bone-like tissues were successfully obtained in vitro, however, after implantation the created tissues lack of a functional vascular supply, resulting in necrotic cores. During the last decade, approaches based in endochondral ossification (ECO) have been increasingly explored. The secretion of osteogenic and angiogenic factors by the cells present in the hypertrophic cartilage templates allows bone tissue repair but also helps tissue vascularization. In chapter I, the most recent ECO approaches used in bone TE are highlighted and discussed. Chapter II describes the methods used to develop an in vitro ECO approach. In chapter III, the results obtained related with the characterization of the obtained bone-like microtissues are presented and discussed. The developed in vitro ECO model relies in the co-culture of 3D micro-cartilaginous templates with umbilical cord-derived mesenchymal stem/stromal cells (UCMSCs) and human umbilical vein endothelial cells (HUVECs). Our hypothesis is that such engineered and privileged microenvironment would mimic the ECO process, leading to the in vitro production of vascularized bone-like microtissues. For that, MSCs-only 3D microtemplates were produced at high-rates and cultured in vitro for 21 days. Then, such microtemplates were co-cultured with UCMSCs and HUVECs within liquefied and multilayered microcapsules, in dynamic conditions, for another 21 days. Microcapsules with chondrogenically-primed 3D microtemplates were cultured with (ECO) and without (ECO control) osteogenic differentiation factors. Also, microcapsules with non-primed 3D microtemplates were cultured with (IMO) and without (negative control) osteogenic differentiation factors. Results show that both 3D microtemplates and microcapsules were able to maintain cell viability up to 21 days. The cartilaginous nature of the 3D templates cultured in chondrogenic medium was confirmed. After 21 days of encapsulation, the ECO microcapsules presented evidence of bone extracellular matrix production and higher matrix mineralization, being the only condition to present a calcium/phosphorous (Ca/P) ratio (1.71) close to the native hydroxyapatite ratio (1.67). Furthermore, both ECO and ECO control conditions successfully induced endothelial cell recruitment. These data show the relevance of using chondrogenically primed 3D microtemplates in bone repair, highlighting the advantage of ECO over IMO approach. In conclusion, this ECO bioencapsulation approach revealed to be a promising bone regeneration strategy. Ultimately, chapter IV discusses the main conclusions and future perspectives related to the potential of ECO approaches in tissue engineering and regenerative medicine.