Controlled Microchannelling in Dense Collagen Scaffolds by Soluble Phosphate Glass Fibers
Showan N. Nazhat, Ensanya A. Abou Neel, Asmeret Kidane, Ifty Ahmed, Chris Hope, Matt Kershaw, Peter D. Lee, Eleanor Stride, Nader Saffari, Jonathan C. Knowles, and Robert A. Brown
Biomacromolecules 2007, 8, 543-551
This technical paper discusses three issues regarding scaffolds: strength, cell viability and formation of microchannels. One of the main issues in tissue engineering design is diffusion of nutrients to cells in dense 3D scaffolds. Utilizing fiber formation technology, this group unidirectionally aligned phosphate glass fibers (PGF) within a 30-40 um diameter in a collagen I matrix, compressed the gel into a sheet, and then rolled the sheet into a spirally assembled scaffold. This technique was performed with or without human oral fibroblasts to determine cell viability under compression.
The phosphate glass fiber scaffold (without cells) underwent mechanical testing. Using a densely packed collagen spiral scaffold as a control, it was shown that a scaffold containing 30% PGF increased Young's Modulus 100x and deformed little prior to failure
In addition, cell viability was determined to be approximately 80% in 30% PGF compressed sheets. Live cells were visualized using calcein AM in the green channel while propidium iodide showed dead cells in the red channel. Confocal laser microscopy captured the images. More death was seen in the spiral scaffold as compared to the sheet. This may be due to improper nutrient diffusion due to the close packing of collagen and cells. No PGF had degraded to form microchannels at that point. In addition, the cells had no preference for PGF or collagen.
To measure degradation of the channels, ion chromatography was utilized. Note, in the figures, that there is mainly a linear trend in the first 6 hours for PGF degradation, and the scaffolds containing more PGFs had a steeper slope. Ultrasound imaging was then used to confirm channel formation. Both microbubbles and dH2O were forced through the scaffold. Ultrasound imaging confirmed microchannel formation on several planes for the PGF degraded scaffolds. However, for the spiral scaffold with only collagen, no microchannels were formed.
Thus, this group demonstrated the potential for possible vascularization of a densely packed scaffold and the use of PGFs as a way to increase mechanical strength. However, to ensure QA, the group needs to refine the technique so that all the fibers remain aligned as this scaffold will probably be utilized "off the shelf". In addition, a worthy future experiment would be to monitor degradation of PGFs within a cell-containing scaffold. It needs to be confirmed that cells remain viable while microchannels are formed. In addition, they could run this experiment for time points longer than 24 hours.
5 comments:
what is ion chromatography?
Ion chromatography basically utilizes the inherent ionic charge molecules to separate them out for analysis. There is normally a stationary and mobile phase. The stationary phase retains your molecule of interest while moving through the column. This is the difference between cation and anion exchange chromatography. For a negatively charged species, like a phosphate, the stationary phase would use a positively charged molecule to retain the phosphate (anion chromatography). Cation chromatography is just the opposite. The calcium ions in this study were retained with a negatively charged stationary phase. The mobile phase is typically an aqueous buffer that carries the solution to the column. As the names suggest, the stationary phase moves much slower than the mobile phase. Two different chromatography experiments were run as there was both an anion and cation quantification.
Based on citation, this group has 2 collaborating PI's that appear to be very interested in the use of biodegradable glass fibers for scaffold strength and potential vascularization. They've been publishing papers with regards to compatibility between cells and the fibers, degradation of the fibers, and effects of the release. However, just doing a quick search on PubMed really only showed groups utilizing bioactive glass (BG) for structural and cell adherent properties in scaffolds. So, not necessarily nutrient diffusion but hopefully groups are working on it and they just haven't published yet.
Were the cells seeded into the microchannels after the completed creation of the scaffold or while the scaffold is being formed? In the bioE118 design project, my group also designed a micro-channeled scaffold as well. In slight contrast, however, we created microgrooves in PLLA scaffolds and seeded bone marrow stromal cells in an attempt to regenerate ACL. Was there any data/results on the change in mechanical properties of the scaffold as the PGF degraded?
The cells were actually never seeded into the microchannels(MC). The MC's were made through the degradation of the phosphate glass fibers and the cells surround them because they happened to be in the collagen I when it set. So, the cells line the channels but are a part of the channel wall with the collagen. In response to the mechanical properties, they were more concerned with the proper degradation of the fibers to form microchannels, so they monitored the degradation rate and tested for perfusion through the scaffold. However, the paper does follow previous work using phosphate glass fibers and shows the mechanical strength before degradation with the fibers entact. I'm sure the group was so happy to get a positive result with the channels that they didn't really look into the strength after degradation. Perhaps the next paper as this group is setting a trend with PGFs...
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