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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.


Quantification of DNA, GAG, and HYP deposition on the co-cultured scaffolds. The scaffolds were sectioned into equal thirds and analyzed separately. All data are mean values plus standard deviations. Groups were tested with n = 4 replicates. (a) DNA amounts were not significantly different between the scaffold regions, but it did significantly increase from day 1 and then stayed relatively constant. (b) There was a significantly higher deposition for GAG/DNA in the FB region compared to the OB region for days 14 and 28, with the transition region having intermediate values between the two. (c) There were no significant differences between groups for HYP/DNA deposition, but there was significantly increasing collagen content over time. Continually increasing collagen deposition is indicative of active and viable cells on the scaffold, in this co-culture model.GAG: glycosaminoglycan; HYP: hydroxyproline; FB: fibroblast; OB: osteoblast.*Statistical significance of p < 0.05.
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fig7-2041731414542294: Quantification of DNA, GAG, and HYP deposition on the co-cultured scaffolds. The scaffolds were sectioned into equal thirds and analyzed separately. All data are mean values plus standard deviations. Groups were tested with n = 4 replicates. (a) DNA amounts were not significantly different between the scaffold regions, but it did significantly increase from day 1 and then stayed relatively constant. (b) There was a significantly higher deposition for GAG/DNA in the FB region compared to the OB region for days 14 and 28, with the transition region having intermediate values between the two. (c) There were no significant differences between groups for HYP/DNA deposition, but there was significantly increasing collagen content over time. Continually increasing collagen deposition is indicative of active and viable cells on the scaffold, in this co-culture model.GAG: glycosaminoglycan; HYP: hydroxyproline; FB: fibroblast; OB: osteoblast.*Statistical significance of p < 0.05.

Mentions: The last objective of this study was to quantify the basic components of the ECM deposited on the static scaffolds in the FB, OB, and transition regions. These components are GAGs and collagen measured by Alcian Blue staining and HYP content, respectively. Deposited matrix distribution was measured for three regions of each scaffold: the FB, transition, and OB regions. The DNA quantified in Figure 7 indicated no significant differences between particular sections representing uniform cell dispersal over the scaffold, including the transition section. There was a significant increase in DNA after day 1 and the cells seemed to plateau between days 7 and 14. DNA was used to normalize the GAG and HYP data. GAG/DNA measurement was significantly higher in the FB region compared to the other regions on days 14 and 28. No significant collagen deposition difference between scaffold regions was determined in these static conditions. A continual and significantly increasing deposition of collagen over the entire 28-day study was found. This indicates good cell viability and activity on the scaffold. Even though not statistically significant, an interesting observation was that the ECM detected in the transition region had intermediate values in between the OB and FB regions, with the exception of day 28 for the collagen assessment, and would suggest mixed contributions from each cell type on the scaffold. Further testing in future studies would be needed to prove this hypothesis.


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

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

Quantification of DNA, GAG, and HYP deposition on the co-cultured scaffolds. The scaffolds were sectioned into equal thirds and analyzed separately. All data are mean values plus standard deviations. Groups were tested with n = 4 replicates. (a) DNA amounts were not significantly different between the scaffold regions, but it did significantly increase from day 1 and then stayed relatively constant. (b) There was a significantly higher deposition for GAG/DNA in the FB region compared to the OB region for days 14 and 28, with the transition region having intermediate values between the two. (c) There were no significant differences between groups for HYP/DNA deposition, but there was significantly increasing collagen content over time. Continually increasing collagen deposition is indicative of active and viable cells on the scaffold, in this co-culture model.GAG: glycosaminoglycan; HYP: hydroxyproline; FB: fibroblast; OB: osteoblast.*Statistical significance of p < 0.05.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

fig7-2041731414542294: Quantification of DNA, GAG, and HYP deposition on the co-cultured scaffolds. The scaffolds were sectioned into equal thirds and analyzed separately. All data are mean values plus standard deviations. Groups were tested with n = 4 replicates. (a) DNA amounts were not significantly different between the scaffold regions, but it did significantly increase from day 1 and then stayed relatively constant. (b) There was a significantly higher deposition for GAG/DNA in the FB region compared to the OB region for days 14 and 28, with the transition region having intermediate values between the two. (c) There were no significant differences between groups for HYP/DNA deposition, but there was significantly increasing collagen content over time. Continually increasing collagen deposition is indicative of active and viable cells on the scaffold, in this co-culture model.GAG: glycosaminoglycan; HYP: hydroxyproline; FB: fibroblast; OB: osteoblast.*Statistical significance of p < 0.05.
Mentions: The last objective of this study was to quantify the basic components of the ECM deposited on the static scaffolds in the FB, OB, and transition regions. These components are GAGs and collagen measured by Alcian Blue staining and HYP content, respectively. Deposited matrix distribution was measured for three regions of each scaffold: the FB, transition, and OB regions. The DNA quantified in Figure 7 indicated no significant differences between particular sections representing uniform cell dispersal over the scaffold, including the transition section. There was a significant increase in DNA after day 1 and the cells seemed to plateau between days 7 and 14. DNA was used to normalize the GAG and HYP data. GAG/DNA measurement was significantly higher in the FB region compared to the other regions on days 14 and 28. No significant collagen deposition difference between scaffold regions was determined in these static conditions. A continual and significantly increasing deposition of collagen over the entire 28-day study was found. This indicates good cell viability and activity on the scaffold. Even though not statistically significant, an interesting observation was that the ECM detected in the transition region had intermediate values in between the OB and FB regions, with the exception of day 28 for the collagen assessment, and would suggest mixed contributions from each cell type on the scaffold. Further testing in future studies would be needed to prove this hypothesis.

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.