Electronic Thesis and Dissertation Repository

Degree

Doctor of Philosophy

Program

Chemical and Biochemical Engineering

Collaborative Specialization

Musculoskeletal Health Research

Supervisor

Flynn, Lauren

2nd Supervisor

Gillies, Elizabeth

Co-Supervisor

Abstract

The development of in vitro cell culture models that investigate tissue-specific effects of the extracellular matrix (ECM) on stem/progenitor cell lineage-commitment can contribute towards the design of improved cell delivery strategies. This thesis developed processing methods that conserved ECM bioactivity to generate well-characterized 2- and 3-D culture platforms that facilitated the evaluation of ECM composition on the adipogenic and osteogenic differentiation of human adipose-derived stromal cells (ASCs). Initial work compared α-amylase and pepsin digestion as methods to fabricate ECM coatings. The effects of enzyme processing and ECM composition were explored using human decellularized adipose tissue (DAT) and bovine tendon collagen as matrix sources. The α-amylase-digested coatings were softer and more stable, with a complex composition and fibrillar architecture. ASCs cultured on α-amylase-digested ECM retained a spindle-shaped morphology, with enhanced proliferation on the α-amylase-digested DAT. Further, the α-amylase-digested DAT enhanced adipogenesis, based on adipogenic gene expression, glycerol-3-phosphate dehydrogenase (GPDH) enzyme activity, and perilipin staining under differentiation conditions. To further evaluate the effects of tissue-specific ECM composition on ASC differentiation, bovine trabecular bone was explored as a compositionally distinct ECM source. A detergent-free protocol was developed for obtaining decellularized trabecular bone (DTB). Immunohistochemical and biochemical techniques were used to compare the composition of the DTB and DAT, demonstrating higher levels of glycosaminoglycans in the DTB and enhanced expression of basement membrane proteins (collagen IV, laminin, collagen VI) in the DAT. To investigate the potential of applying a tissue-specific approach within a 3-D culture system, cryo-milled DAT or DTB particles were incorporated within methacrylated chondroitin sulphate (MCS) hydrogels. ASC viability, adipogenesis and osteogenesis were assessed in the MCS+DAT, MCS+DTB and MCS alone. The findings indicated that the incorporation of DAT provided an adipo-conductive microenvironment, as seen by enhanced adipogenic gene expression, GPDH enzyme activity and intracellular lipid accumulation under differentiation conditions. The preliminary osteogenic data suggested that the DTB may have osteo-inductive effects, as seen by early stage osteogenic gene expression (OPN and ON) under proliferation conditions. ii Overall, this thesis provided a body of evidence supporting that tissue-specific ECM composition can be harnessed in biomaterials design to promote the lineage-specific differentiation of ASCs.

Download

Included in

Biomaterials Commons

Share

COinS