Lack of vascularization has been suggested as one of the major limitations for current
tissue-engineered (TE) skin substitutes. Until vascularization of the implanted tissue
occurs, nutrients delivery and waste removal still rely primarily on passive diffusion. As
a result, it is now widely accepted that TE skin substitutes should contain integrated
vasculature before transplantation to improve their survival in vivo.
In this dissertation, an in vitro model of TE skin substitute was developed with integrated
flow networks in a perfusion bioreactor. First, a new approach was suggested based on
centrifugation for obtaining highly concentrated yet porous collagen-glycosaminoglycan
scaffolds. Water uptake, morphology, mechanical properties, and tissue-engineering
potential of the concentrated scaffolds were investigated. The results show that the new
approach can lead to scaffolds containing four times as much as collagen as that in
conventional unconcentrated scaffolds. Water uptake and mechanical properties were significantly improved. In addition, well-stratified TE skin substitutes were obtained
using the concentrated scaffolds under static culture conditions.
A perfusion bioreactor system was designed for TE skin substitutes with integrated flow
networks. The perfusion bioreactor provided not only a submerged culture mode for
keratinocyte proliferation but also an air-liquid interface culture mode for keratinocyte
differentiation. Utilizing the perfusion bioreactor, TE skin substitutes with integrated
flow networks were successfully fabricated in vitro. The effect of flow on the epidermis
formation was assessed through histology, immunostaining, and tissue viability. TE skin
substitutes cultured at 1000 μL/h perfusion rate showed a well-stratified epidermis, along
with anatomy comparable with that of control but thicker stratum spinosum and stratum
corneum.
Finally, to obtain large 3D organ/tissue substitutes, an adhesion technique was developed
based on albumin/glutaraldehyde bioadhesives to bond collagen scaffolds. Mechanical
properties of adhesion were investigated, and the results suggest that the bioadhesives
bonded collagen scaffolds very well. Biocompatibility of the bioadhesives was
demonstrated in vitro. It is feasible to use the bioadhesives to obtained closed flow
networks in a collagen composite. During this investigation, interference in a commonly
used cell viability assay due to adhesive components was discovered.