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Co-cultured tissue-specific scaffolds for tendon/bone interface engineering.

Cooper JO, Bumgardner JD, Cole JA, Smith RA, Haggard WO - J Tissue Eng (2014)

Bottom Line: Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding.Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions.In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The University of Memphis, Memphis, TN, USA.

ABSTRACT
The tendon/ligament-to-bone interface has a complex organization to enable transfer of forces through the tendon/ligament to the bone. The purpose of this study is to create a co-culture environment enabling a tissue-specific tendon region and tissue-specific bone region on a degradable scaffold, using NIH 3T3 fibroblast-deposited extracellular matrix and MC 3T3 osteoblast-deposited extracellular matrix, respectively. Before full characterization of the deposited extracellular matrix coating can be analyzed, co-culture parameters including culture medium and seeding technique should be addressed. An appropriate medium formulation was developed to reduce fibroblast to osteoblast mineralization by adjusting beta-glycerophosphate concentrations. Standard growth medium with fetal bovine serum + 3 mM beta-glycerophosphate + 25 µg/mL ascorbic acid was found to be the most suitable formulation evaluated in these study conditions. Seeding and cell migration studies of co-cultured fibroblast- and osteoblast-specific scaffolds were performed to identify whether tissue regions could be created on the scaffold. Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding. Finally, a preliminary analysis of basic extracellular matrix components was measured in the fibroblast, osteoblast, and transition regions. Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions. In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold.

No MeSH data available.


Related in: MedlinePlus

Merged fluorescent images over a 42-h period. Images show that the cells maintain the tissue-specific regions on the scaffold. No noticeable migration was observed in the measured time frame. There are green-labeled FB in the OB region and red-labeled OB in the FB region, even though no major cell migration was observed.FB: fibroblast; OB: osteoblast.
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fig6-2041731414542294: Merged fluorescent images over a 42-h period. Images show that the cells maintain the tissue-specific regions on the scaffold. No noticeable migration was observed in the measured time frame. There are green-labeled FB in the OB region and red-labeled OB in the FB region, even though no major cell migration was observed.FB: fibroblast; OB: osteoblast.

Mentions: The second objective of this study was to investigate seeding a single scaffold in co-culture to create FB- and OB-specific regions. The FBs were stained with green tracking probe and the OBs were labeled with a red tracking probe. The montage image in Figure 4 shows the entire 10 mm × 60mm scaffold first with the green filter enabled then with the red filter. Each montage comprises approximately 200 individual 4× magnification images taken with the aid of a motorized stage and stitched together using BIOQUANT software. As can be seen in Figure 4, both FBs and OBs are attached on their respective half of the scaffold creating the tissue-specific regions. The general tissue-specific regions at low magnification are easier to distinguish. Figure 5 contains the two non-merged images with smaller selected regions shown at higher magnifications. There are very few FBs in the OB region and vice versa. On the left-hand edge of the OB side, FBs did attach and are more evident at higher exposure times. However, most cells are located in their respective regions with a decreasing gradient across the scaffold. There was no noticeable migration of cells between regions observed over the 42 h in Figure 6. The fluorescence label loses intensity over time, and therefore, the exposure time was increased by the 42-h image to intensify the colors on the scaffold. Because of the increased exposure, the 42-h image has a noticeable amount of unanticipated autofluorescence in the periphery of the image. This is due to the cyanoacrylate used to make the custom glass cover slip Petri dish for imaging and not the FB fluorescing.


Co-cultured tissue-specific scaffolds for tendon/bone interface engineering.

Cooper JO, Bumgardner JD, Cole JA, Smith RA, Haggard WO - J Tissue Eng (2014)

Merged fluorescent images over a 42-h period. Images show that the cells maintain the tissue-specific regions on the scaffold. No noticeable migration was observed in the measured time frame. There are green-labeled FB in the OB region and red-labeled OB in the FB region, even though no major cell migration was observed.FB: fibroblast; OB: osteoblast.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2 - License 3
Show All Figures
getmorefigures.php?uid=PMC4221948&req=5

fig6-2041731414542294: Merged fluorescent images over a 42-h period. Images show that the cells maintain the tissue-specific regions on the scaffold. No noticeable migration was observed in the measured time frame. There are green-labeled FB in the OB region and red-labeled OB in the FB region, even though no major cell migration was observed.FB: fibroblast; OB: osteoblast.
Mentions: The second objective of this study was to investigate seeding a single scaffold in co-culture to create FB- and OB-specific regions. The FBs were stained with green tracking probe and the OBs were labeled with a red tracking probe. The montage image in Figure 4 shows the entire 10 mm × 60mm scaffold first with the green filter enabled then with the red filter. Each montage comprises approximately 200 individual 4× magnification images taken with the aid of a motorized stage and stitched together using BIOQUANT software. As can be seen in Figure 4, both FBs and OBs are attached on their respective half of the scaffold creating the tissue-specific regions. The general tissue-specific regions at low magnification are easier to distinguish. Figure 5 contains the two non-merged images with smaller selected regions shown at higher magnifications. There are very few FBs in the OB region and vice versa. On the left-hand edge of the OB side, FBs did attach and are more evident at higher exposure times. However, most cells are located in their respective regions with a decreasing gradient across the scaffold. There was no noticeable migration of cells between regions observed over the 42 h in Figure 6. The fluorescence label loses intensity over time, and therefore, the exposure time was increased by the 42-h image to intensify the colors on the scaffold. Because of the increased exposure, the 42-h image has a noticeable amount of unanticipated autofluorescence in the periphery of the image. This is due to the cyanoacrylate used to make the custom glass cover slip Petri dish for imaging and not the FB fluorescing.

Bottom Line: Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding.Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions.In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, The University of Memphis, Memphis, TN, USA.

ABSTRACT
The tendon/ligament-to-bone interface has a complex organization to enable transfer of forces through the tendon/ligament to the bone. The purpose of this study is to create a co-culture environment enabling a tissue-specific tendon region and tissue-specific bone region on a degradable scaffold, using NIH 3T3 fibroblast-deposited extracellular matrix and MC 3T3 osteoblast-deposited extracellular matrix, respectively. Before full characterization of the deposited extracellular matrix coating can be analyzed, co-culture parameters including culture medium and seeding technique should be addressed. An appropriate medium formulation was developed to reduce fibroblast to osteoblast mineralization by adjusting beta-glycerophosphate concentrations. Standard growth medium with fetal bovine serum + 3 mM beta-glycerophosphate + 25 µg/mL ascorbic acid was found to be the most suitable formulation evaluated in these study conditions. Seeding and cell migration studies of co-cultured fibroblast- and osteoblast-specific scaffolds were performed to identify whether tissue regions could be created on the scaffold. Fibroblast and osteoblast regions were successfully seeded and little to no cell migration was observed up to 42 h after seeding. Finally, a preliminary analysis of basic extracellular matrix components was measured in the fibroblast, osteoblast, and transition regions. Tissue-specific DNA, glycosaminoglycan, and collagen were found in uniform amounts on the scaffolds and were not different significantly between scaffold regions. In conclusion, initial steps to create tissue-specific fibroblast and osteoblast regions on a degradable scaffold were successful in preparation for further characterization investigations as a tendon-to-bone interface scaffold.

No MeSH data available.


Related in: MedlinePlus