Use of a 3D perfusion bioreactor with osteoblasts and osteoblast/endothelial cell co-cultures to improve tissue-engineered bone

The delivery of oxygen, nutrients, and the removal of waste are essential for cellular survival. Culture systems for 3D bone tissue engineering have addressed this issue by utilizing perfusion flow bioreactors that stimulate osteogenic activity through the delivery of oxygen and nutrients by low-shear fluid flow. It is also well established that bone responds to mechanical stimulation, but may desensitize under continuous loading. While perfusion flow and mechanical stimulation are used to increase cellular survival in vitro, 3D tissue-engineered constructs face additional limitations upon in vivo implantation. As it requires significant amounts of time for vascular infiltration by the host, implants are subject to an increased risk of necrosis. One solution is to introduce tissue-engineered bone that has been pre-vascularized through the co-culture of osteoblasts and endothelial cells on 3D constructs. It is unclear from previous studies: 1) how 3D bone tissue constructs will respond to partitioned mechanical stimulation, 2) how gene expression compares in 2D and in 3D, 3) how co-cultures will affect osteoblast activity, and 4) how perfusion flow will affect co-cultures of osteoblasts and endothelial cells. We have used an integrated approach to address these questions by utilizing mechanical stimulation, perfusion flow, and a co-culture technique to increase the success of 3D bone tissue engineering. We measured gene expression of several osteogenic and angiogenic genes in both 2D and 3D (static culture and mechanical stimulation), as well as in 3D cultures subjected to perfusion flow, mechanical stimulation and partitioned mechanical stimulation. Finally, we co-cultured osteoblasts and endothelial cells on 3D scaffolds and subjected them to long-term incubation in either static culture or under perfusion flow to determine changes in gene expression as well as histological measures of osteogenic and angiogenic activity. We discovered that 2D and 3D osteoblast cultures react differently to shear stress, and that partitioning mechanical stimulation does not affect gene expression in our model. Furthermore, our results suggest that perfusion flow may rescue 3D tissue-engineered constructs from hypoxic-like conditions by reducing hypoxia-specific gene expression and increasing histological indices of both osteogenic and angiogenic activity. Future research to elucidate the mechanisms behind these results may contribute to a more mature bone-like structure that integrates more quickly into host tissue, increasing the potential of bone tissue engineering.

USING INDICATORS OF BIOTIC INTEGRITY FOR ASSESSMENT OF STREAM CONDITION

Multiple indices of biotic integrity and biological condition gradient models have been developed and validated to assess ecological integrity in the Laurentian Great Lakes Region. With multiple groups such as Tribal, Federal, and State agencies as well as scientists and local watershed management or river-focused volunteer groups collecting data for bioassessment it is important that we determine the comparability of data and the effectiveness of indices applied to these data for assessment of natural systems. We evaluated the applicability of macroinvertebrate and fish community indices for assessing site integrity. Site quality (i.e., habitat condition) could be classified differently depending on which index was applied. This highlights the need to better understand the metrics driving index variation as well as reference conditions for effective communication and use of indices of biotic integrity in the Upper Midwest. We found the macroinvertebrate benthic community index for the Northern Lakes and Forests Ecoregion and a coldwater fish index of biotic integrity for the Upper Midwest were most appropriate for use in the Big Manistee River watershed based on replicate sampling, ability to track trends over time and overall performance. We evaluated three sites where improper road stream crossings (culverts) were improved by replacing them with modern full-span structures using the most appropriate fish and macroinvertebrate IBIs. We used a before-after-control-impact paired series analytical design and found mixed results, with evidence of improvement in biotic integrity based on macroinvertebrate indices at some of the sites while most sites indicated no response in index score. Culvert replacements are often developed based on the potential, or the perception, that they will restore ecological integrity. As restoration practitioners, researchers and managers, we need to be transparent in our goals and objectives and monitor for those results specifically. The results of this research serve as an important model for the broader field of ecosystem restoration and support the argument that while biotic communities can respond to actions undertaken with the goal of overall restoration, practitioners should be realistic in their expectations and claims of predicted benefit, and then effectively evaluate the true impacts of the restoration activities.