ABSTRACT
Autologous fat grafting is hampered by unpredictable outcomes due to high tissue resorption. Hydrogels based on enzymatically pretreated tunicate nanocellulose (ETC) and alginate (ALG) are biocompatible, safe, and present physiochemical properties capable of promoting cell survival. Here, we compared in situ and ex situ crosslinking of ETC/ALG hydrogels combined with lipoaspirate human adipose tissue (LAT) to generate an injectable formulation capable of retaining dimensional stability in vivo. We performed in situ crosslinking using two different approaches; inducing Ca2+ release from CaCO3 microparticles (CMPs) and physiologically available Ca2+ in vivo. Additionally, we generated ex situ-crosslinked, 3D-bioprinted hydrogel-fat grafts. We found that in vitro optimization generated a CMP-crosslinking system with comparable stiffness to ex situ-crosslinked gels. Comparison of outcomes following in vivo injection of each respective crosslinked hydrogel revealed that after 30 days, in situ crosslinking generated fat grafts with less shape retention than 3D-bioprinted constructs that had undergone ex situ crosslinking. However, CMP addition improved fat-cell distribution and cell survival relative to grafts dependent on physiological Ca2+ alone. These findings suggested that in situ crosslinking using CMP might promote the dimensional stability of injectable fat-hydrogel grafts, although 3D bioprinting with ex situ crosslinking more effectively ensured proper shape stability in vivo.
ABSTRACT
Establishing a vascular network in biofabricated tissue grafts is essential for ensuring graft survival. Such networks are dependent on the ability of the scaffold material to facilitate endothelial cell adhesion; however, the clinical translation potential of tissue-engineered scaffolds is hindered by the lack of available autologous sources of vascular cells. Here, we present a novel approach to achieving autologous endothelialisation in nanocellulose-based scaffolds by using adipose tissue-derived vascular cells on nanocellulose-based scaffolds. We used sodium periodate-mediated bioconjugation to covalently bind laminin to the scaffold surface and isolated the stromal vascular fraction and endothelial progenitor cells (EPCs; CD31+CD45-) from human lipoaspirate. Additionally, we assessed the adhesive capacity of scaffold bioconjugationin vitrousing both adipose tissue-derived cell populations and human umbilical vein endothelial cells. The results showed that the bioconjugated scaffold exhibited remarkably higher cell viability and scaffold surface coverage by adhesion regardless of cell type, whereas control groups comprising cells on non-bioconjugated scaffolds exhibited minimal cell adhesion across all cell types. Furthermore, on culture day 3, EPCs seeded on laminin-bioconjugated scaffolds showed positive immunofluorescence staining for the endothelial markers CD31 and CD34, suggesting that the scaffolds promoted progenitor differentiation into mature endothelial cells. These findings present a possible strategy for generating autologous vasculature and thereby increase the clinical relevance of 3D-bioprinted nanocellulose-based constructs.
