3D Spheroid Models for Functional Evaluation of Endothelial Cell Differentiation
Objectives: Establishing angiogenesis is a key factor in regenerative medicine and the future of periodontal tissue engineering. Unfortunately, clinical applications of tissue engineering are limited by the lack of adequate blood supply. Endothelial cells are a vital component of the capillaries that provide adequate blood supply and excretion of wastes in tissues. The aim of the study was to examine the efficacy of 3D endothelial spheroid culture systems. We hypothesized that human adipose-derived stem cells (hASCs) would more efficiently differentiate toward endothelial lineage when formed as spheroids. Methods: hASCs isolated from elective liposuction aspirates under an IRB-approved protocol were seeded in varying concentrations of either collagen (2, 6mg/mL) or collagen-ELP (1:3 mass ratio) hydrogels (40,000cells/hydrogel). Some hydrogels were cross-linked utilizing carbodiimide chemistry. Cells were cultured in endothelial differentiation medium (EGM-2-MV) and collected on day 0 and 21. Assays for viability, DNA content, total protein content, and for endothelial differentiation markers (von Willebrand factor, CD31, Low-density lipoprotein uptake) were performed. Results: Cells remained viable (>70%) in all hydrogels. Cells cultured in 2mg/mL collagen and 2:6 collagen-ELP showed spread morphology and proliferated with increase in DNA and protein content (Table 1). Cells cultured in all other hydrogels revealed spheroidal morphology with either no change (due to contact-inhibited growth arrest) or decrease (due to cell death) in DNA content. Although all hydrogels showed expression of endothelial markers, spheroid formation generally correlated with higher expression. Conclusions: Our hydrogels support spheroid formation and endothelial cell differentiation; mimicking physiological conditions in which neo-angiogenesis can occur. Conditions displaying higher endothelial marker expression may indicate benefit of spheroid formation. Further studies aim to elucidate how hydrogel stiffness may affect spheroid formation by comparing them against scaffold-free ELP-polyelectrolyte coating that also form spheroids. We will also explore co-culturing strategies to further enhance endothelial differentiation in this new promising approach.
Division: AADR/CADR Annual Meeting
Meeting:2016 AADR/CADR Annual Meeting (Los Angeles, California) Location: Los Angeles, California
Year: 2016 Final Presentation ID:0164 Abstract Category|Abstract Category(s):Dental Materials 2:Polymer-based Materials
Authors
Clark, Kendra
( University of Mississippi Medical Center
, Jackson
, Mississippi
, United States
)
Janorkar, Amol
( University of Mississippi Medical Center
, Jackson
, Mississippi
, United States
)
Support Funding Agency/Grant Number: NIH/NIDCR R03DE024257
Financial Interest Disclosure: NONE
Hydrogels that form 3D spheroidal structures of hASCs show enhanced endothelial differentiation.
DNA
Protein
Von Willebrand Factor
CD 31
Composition of Hydrogels
Hydrogel Morphology
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
Day 0
Day 21
2 mg/mL Collagen
Spread
1.31 ± 1.90
4.61 ± 1.21*
53.42 ± 3.22
396.16 ± 61.22*
25.20 ± 3.21
0.00 ± 0.00*
47.41 ± 15.94
0.00 ± 0.00*
2 mg/mL Collagen crosslinked with EDC/NHS
Spheroidal
1.84 ± 0.98
1.13 ± 0.52†
69.04 ± 7.64†
159.04 ± 17.88*†
10.64 ± 3.55†
19.40 ± 5.60†
31.03 ± 8.62
0.00 ± 0.00*
2:6 mg/mL Collagen-ELP
Spread
1.31 ± 0.48
1.99 ± 0.70†
85.07 ± 35.49
292.60 ± 15.40*†
14.63 ± 3.75†
2.10 ± 0.00*†
24.60 ± 9.94
0.00 ± 0.00*
2:6 mg/mL Collagen-ELP crosslinked with EDC/NHS
Spheroidal
2.38 ± 0.80
1.43 ± 0.84†
58.77 ± 9.39
115.48 ± 36.08*†
6.09 ± 1.88†
19.16 ± 4.52*†
14.76 ± 6.82†
33.99 ± 20.94†
6 mg/mL Collagen
Spheroidal
2.14 ± 1.07
0.66 ± 0.32*†
77.26 ± 10.28†
50.96 ± 8.17*†
6.32 ± 2.51†
41.84 ± 10.10*†
10.12 ± 10.41†
54.57 ± 17.77*†
6 mg/mL Collagen crosslinked with EDC/NHS
Spheroidal
1.82 ± 1.16
0.41 ± 0.17*†
44.79 ± 4.26†
51.78 ± 6.94†
8.11 ± 4.06†
73.97 ± 14.10*†
9.25 ± 3.48†
0.00 ± 0.00*
Cells cultured in various compositions of hydrogels were collected at 0 and 21 days. Cells were lysed and analyzed in triplicate for DNA and protein content, and von willebrand factor and CD31 endothelial marker expression. Values are listed as the average (µg) ± 95% confidence interval. Statistical analysis performed by ANOVA. * is indicative of p<0.05 comparing 0 and 21 days of each hydrogel. † indicates p<0.05 compared to 2 mg/mL collagen hydrogels. n=3. 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC)/N-Hydroxysuccinimide (NHS).