Abstract
Hyperglycemia-mediated, nonenzymatic collagen cross-links such as pentosidine (PENT) can have deleterious effects on cellular interactions with the extracellular matrix (ECM). Present techniques to quantify PENT are limited, motivating the need for improved methods to study the accumulation and contribution of PENT toward diabetic clinical challenges such as impaired bone healing. Current methods for studying PENT are destructive, laborious, and frequently employ oversimplified collagen films that lack the complexity of the native ECM. The primary goal of this study was to evaluate the capacity of time-resolved fluorescence spectroscopy (TRFS) to detect PENT in cell-secreted ECMs possessing enhanced compositional complexity. To demonstrate an application of this method, we assessed the response of human mesenchymal stem cells (MSCs) to cross-linked substrates to explore the role of detected PENT on osteogenic differentiation. We exposed MSC-secreted decellularized matrices (DMs) to 0.66 M ribose for 2 weeks and used TRFS to detect the accumulation of PENT. Ribose treatment resulted in a 30 nm blue shift in peak fluorescence emission and a significant decrease in average lifetime compared to that of control DMs (4.4 ± 0.3 ns vs 3.5 ± 0.09 ns). Evaluation of samples with high performance liquid chromatography (HPLC) confirmed that changes in observed fluorescence were due to PENT accumulation. A strong correlation was found between TRFS parameters and the HPLC measurement of PENT, validating the use of TRFS as an alternative method of PENT detection. Osteoblastic gene expression was significantly reduced in MSCs seeded on ribose DMs at days 7 and 14. However, no significant differences in calcium deposition were detected between control and ribose DMs. These data demonstrate the efficacy of nondestructive fluorescence spectroscopy to examine the formation of nonenzymatic collagen cross-links within biomimetic culture platforms and showcase one example where an improved biomimetic substrate can be used to probe cell-ECM interactions in the presence of collagen cross-links.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 1944-1954 |
| Number of pages | 11 |
| Journal | ACS Biomaterials Science and Engineering |
| Volume | 3 |
| Issue number | 9 |
| DOIs | |
| State | Published - Sep 11 2017 |
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Keywords
- collagen cross-links
- decellularized matrices
- fluorescence lifetime
- mesenchymal stem cells
- optical imaging
ASJC Scopus subject areas
- Biomaterials
- Biomedical Engineering
Cite this
Detection of Pentosidine Cross-Links in Cell-Secreted Decellularized Matrices Using Time Resolved Fluorescence Spectroscopy. / Mitra, Debika; Fatakdawala, Hussain; Nguyen-Truong, Michael; Creecy, Amy; Nyman, Jeffry; Marcu, Laura; Leach, Jonathan K.
In: ACS Biomaterials Science and Engineering, Vol. 3, No. 9, 11.09.2017, p. 1944-1954.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Detection of Pentosidine Cross-Links in Cell-Secreted Decellularized Matrices Using Time Resolved Fluorescence Spectroscopy
AU - Mitra, Debika
AU - Fatakdawala, Hussain
AU - Nguyen-Truong, Michael
AU - Creecy, Amy
AU - Nyman, Jeffry
AU - Marcu, Laura
AU - Leach, Jonathan K
PY - 2017/9/11
Y1 - 2017/9/11
N2 - Hyperglycemia-mediated, nonenzymatic collagen cross-links such as pentosidine (PENT) can have deleterious effects on cellular interactions with the extracellular matrix (ECM). Present techniques to quantify PENT are limited, motivating the need for improved methods to study the accumulation and contribution of PENT toward diabetic clinical challenges such as impaired bone healing. Current methods for studying PENT are destructive, laborious, and frequently employ oversimplified collagen films that lack the complexity of the native ECM. The primary goal of this study was to evaluate the capacity of time-resolved fluorescence spectroscopy (TRFS) to detect PENT in cell-secreted ECMs possessing enhanced compositional complexity. To demonstrate an application of this method, we assessed the response of human mesenchymal stem cells (MSCs) to cross-linked substrates to explore the role of detected PENT on osteogenic differentiation. We exposed MSC-secreted decellularized matrices (DMs) to 0.66 M ribose for 2 weeks and used TRFS to detect the accumulation of PENT. Ribose treatment resulted in a 30 nm blue shift in peak fluorescence emission and a significant decrease in average lifetime compared to that of control DMs (4.4 ± 0.3 ns vs 3.5 ± 0.09 ns). Evaluation of samples with high performance liquid chromatography (HPLC) confirmed that changes in observed fluorescence were due to PENT accumulation. A strong correlation was found between TRFS parameters and the HPLC measurement of PENT, validating the use of TRFS as an alternative method of PENT detection. Osteoblastic gene expression was significantly reduced in MSCs seeded on ribose DMs at days 7 and 14. However, no significant differences in calcium deposition were detected between control and ribose DMs. These data demonstrate the efficacy of nondestructive fluorescence spectroscopy to examine the formation of nonenzymatic collagen cross-links within biomimetic culture platforms and showcase one example where an improved biomimetic substrate can be used to probe cell-ECM interactions in the presence of collagen cross-links.
AB - Hyperglycemia-mediated, nonenzymatic collagen cross-links such as pentosidine (PENT) can have deleterious effects on cellular interactions with the extracellular matrix (ECM). Present techniques to quantify PENT are limited, motivating the need for improved methods to study the accumulation and contribution of PENT toward diabetic clinical challenges such as impaired bone healing. Current methods for studying PENT are destructive, laborious, and frequently employ oversimplified collagen films that lack the complexity of the native ECM. The primary goal of this study was to evaluate the capacity of time-resolved fluorescence spectroscopy (TRFS) to detect PENT in cell-secreted ECMs possessing enhanced compositional complexity. To demonstrate an application of this method, we assessed the response of human mesenchymal stem cells (MSCs) to cross-linked substrates to explore the role of detected PENT on osteogenic differentiation. We exposed MSC-secreted decellularized matrices (DMs) to 0.66 M ribose for 2 weeks and used TRFS to detect the accumulation of PENT. Ribose treatment resulted in a 30 nm blue shift in peak fluorescence emission and a significant decrease in average lifetime compared to that of control DMs (4.4 ± 0.3 ns vs 3.5 ± 0.09 ns). Evaluation of samples with high performance liquid chromatography (HPLC) confirmed that changes in observed fluorescence were due to PENT accumulation. A strong correlation was found between TRFS parameters and the HPLC measurement of PENT, validating the use of TRFS as an alternative method of PENT detection. Osteoblastic gene expression was significantly reduced in MSCs seeded on ribose DMs at days 7 and 14. However, no significant differences in calcium deposition were detected between control and ribose DMs. These data demonstrate the efficacy of nondestructive fluorescence spectroscopy to examine the formation of nonenzymatic collagen cross-links within biomimetic culture platforms and showcase one example where an improved biomimetic substrate can be used to probe cell-ECM interactions in the presence of collagen cross-links.
KW - collagen cross-links
KW - decellularized matrices
KW - fluorescence lifetime
KW - mesenchymal stem cells
KW - optical imaging
UR - http://www.scopus.com/inward/record.url?scp=85029455979&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85029455979&partnerID=8YFLogxK
U2 - 10.1021/acsbiomaterials.6b00029
DO - 10.1021/acsbiomaterials.6b00029
M3 - Article
AN - SCOPUS:85029455979
VL - 3
SP - 1944
EP - 1954
JO - ACS Biomaterials Science and Engineering
JF - ACS Biomaterials Science and Engineering
SN - 2373-9878
IS - 9
ER -