Tissue-specific bioscaffolds for adipose-derived stem/stromal cell expansion and delivery

Decellularized adipose tissue (DAT) is a promising biomaterial for soft tissue regeneration, and it provides a highly conducive microenvironment for human adipose-derived stem/stromal cell (ASC) attachment, proliferation, and adipogenesis. This thesis focused on developing techniques to fabricate 3-D bioscaffolds from enzymatically-digested DAT as platforms for ASC culture and delivery in adipose tissue engineering and large-scale ASC expansion. Initial work investigated chemically crosslinked microcarriers fabricated from pepsin-digested DAT as injectable adipo-inductive substrates for ASCs. DAT microcarriers highly supported ASC adipogenesis compared to gelatin microcarriers in a CELLSPIN system, as confirmed by glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, lipid accumulation, and endpoint RT-PCR. ASCs cultured on DAT microcarriers in proliferation medium also had elevated PPARγ, C/EBPα, and LPL expression which suggested adipo-inductive properties. In vivo testing of the DAT microcarriers exhibited stable volume retention and enhanced cellular infiltration, tissue remodeling, and angiogenesis. Building from this work, non-chemically crosslinked porous foams and bead foams were fabricated from α-amylase-digested DAT for soft tissue regeneration. Foams were stable and strongly supported ASC adipogenesis based on GPDH activity and endpoint RT-PCR. PPARγ, C/EBPα, and LPL expression in ASCs cultured on the foams in proliferation media indicated adipo-inductive properties. Foams with Young’s moduli similar to human fat also influenced ASC adipogenesis by enhanced GPDH activity. In vivo adipogenesis accompanied by a potent angiogenic response and rapid resorption showed their potential use in wound healing applications. Finally, non-chemically crosslinked porous microcarriers synthesized from α-amylase-digested DAT were investigated for ASC expansion. DAT microcarriers remained stable in culture and supported significantly higher ASC proliferation compared to Cultispher-S microcarriers in a CELLSPIN system. ASC immunophenotype was preserved for all expanded groups, with reduced adhesion marker expression under dynamic conditions. DAT microcarrier expansion upregulated ASC expression of early adipogenic (PPARγ, LPL) and chondrogenic (COMP) markers without inducing a mature phenotype. DAT microcarrier expanded ASCs also showed similar levels of adipogenesis and osteogenesis compared to Cultispher-S despite a significantly higher population fold-change, and had the highest level of chondrogenesis among all groups. This study demonstrates the promising use of DAT microcarriers as a clinically relevant strategy for ASC expansion while maintaining multilineage differentiation capacity.

Chitosan fibrous scaffolds for cartilage tissue engineering

The biocompatibility of chitosan and its similarity with glycosaminoglycans make it attractive for cartilage engineering despite its limited cell adhesion properties. Structural and chemical characteristics of chitosan scaffolds may be improved for cartilage engineering application. We planned to evaluate chitosan meshes produced by a novel technique and the effect of chitosan structure on mesenchymal stem cells (MSCs) chondrogenesis. Another objective was to improve cell adhesion and chondrogenesis on chitosan by modifying the chemical composition of the scaffold (reacetylation, collagen II, or hyaluronic acid (HA) coating). A replica molding technique was developed to produce chitosan meshes of different fiber-width. A polyglycolic acid (PGA) mesh served as a reference. Constructs were analyzed at two and 21 days after seeding chondrocytes with confocal microscopy, scanning electron microscopy, histology, and quantitative analysis (weights, DNA, glycosaminoglycans, collagen II). Chondrocytes maintained their phenotypic appearance and a high viability but attached preferentially to PGA. Matrix production per chondrocyte was superior on chitosan. Chitosan meshes and sponges were analyzed after seeding and culture of MSCs under chondrogenic condition for 21 days. The cellularity was similar between groups but matrix production was greater on meshes. Chitosan and reacetylated-chitosan scaffolds were coated with collagen II or HA. Scaffolds were characterized prior to seeding MSCs. Chitosan meshes were then coated with collagen at two densities. PGA served as a reference. Constructs were evaluated after seeding or culture of MSCs for 21 days in chondrogenic medium. MSCs adhered less to reacetylated-chitosan despite collagen coating. HA did not affect cell adhesion. The cell attachment on chitosan correlated with collagen density. The cell number and matrix production were improved after culture in collagen coated meshes. The differences between PGA and chitosan are likely to result from the chemical composition. Chondrogenesis is superior on chitosan meshes compared to sponges. Collagen II coating is an efficient way to overcome poor cell adhesion on chitosan. These findings encourage the use of chitosan meshes coated with collagen II and confirm the importance of biomimetic scaffolds for tissue engineering. The decreased cell adhesion on reacetylated chitosan and the poor mechanical stability of PGA limit their use for tissue engineering.

Identification and functional analysis of novel genes expressed in the Anterior Visceral Endoderm

During early vertebrate development, the correct establishment of the body axes is critical. The anterior pole of the mouse embryo is established when Distal Visceral Endoderm (DVE) cells migrate to form the Anterior Visceral Endoderm (AVE). Symmetrical expression of Lefty1, Cer1 and Dkk1 determines the direction of DVE migration and the future anterior side. In addition to the establishment of the Anterior-Posterior axis, the AVE has also been implicated in anterior neural specification. To better understand the role of the AVE in these processes, we have performed a differential screening using Affymetrix GeneChip technology with AVE cells isolated from cer1P-EGFP transgenic mouse embryos. We found 175 genes which were upregulated in the AVE and 36 genes in the Proximal-posterior sample. Using DAVID software, we characterized the AVE cell population regarding cellular component, molecular function and biological processes. Among the genes that were found to be upregulated in the AVE, several novel genes were identified. Four of these transcripts displaying high-fold change in the AVE were further characterized by in situ hybridization in early stages of development in order to validate the screening. From those four selected genes, one, denominated Adtk1, was chosen to be functionally characterized by targeted inactivation in ES cells. Adtk1 encodes for a serine/threonine kinase. Adtk1 null mutants are smaller and present short limbs due to decreased mineralization, suggesting a potential role in chondrogenesis during limb development. Taken together, these data point to the importance of reporting novel genes present in the AVE.