Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells
Abstract
:1. Introduction
2. Results
2.1. Expression of the FGF Receptors 1−4 in Bovine and Human NP Cells
2.2. Expression of NP Cell Markers in Bovine and Human NP Cells
2.3. Gene Expression of IVD Matrix Molecule and Catabolic Markers in Bovine and Human NP Cells
2.4. Proliferation and Matrix Protein Synthesis of Bovine and Human NP Cells
2.5. Histology Staining
3. Discussion
4. Materials and Methods
4.1. Materials and Reagents
4.2. Methods
4.2.1. Cell Isolation and In Vitro Culture in FBG-HA Hydrogel
4.2.2. Biochemical Analysis
4.2.3. Gene Expression Analysis
4.2.4. Safranin O/Fast Green Staining and Immunohistochemistry
4.2.5. Statistical Analysis
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Gene | Primer/Probe Type | Sequence |
---|---|---|
bCOL1 | Primer fw (5′–3′) | TGC AGT AAC TTC GTG CCT AGC A |
Primer rev (5′–3′) | CGC GTG GTC CTC TAT CTC CA | |
Probe (5′FAM/3′TAMRA) | CAT GCC AAT CCT TAC AAG AGG CAA CTG C | |
bCOL2 | Primer fw (5′–3′) | AAG AAA CAC ATC TGG TTT GGA GAA A |
Primer rev (5′–3′) | TGG GAG CCA GGT TGT CAT C | |
Probe (5′FAM/3′TAMRA) | CAA CGG TGG CTT CCA CTT CAG CTA TGG | |
bACAN | Primer fw (5′–3′) | CCA ACG AAA CCT ATG ACG TGT ACT |
Primer rev (5′–3′) | GCA CTC GTT GGC TGC CTC | |
Probe (5′FAM/3′TAMRA) | ATG TTG CAT AGA AGA CCT CGC CCT CCA T | |
bMMP3 | Primer fw (5′–3′) | GGC TGC AAG GGA CAA GGA A |
Primer rev (5′–3′) | CAA ACT GTT TCG TAT CCT TTG CAA | |
Probe (5′FAM/3′TAMRA) | CAC CAT GGA GCT TGT TCA GCA ATA TCT AGA AAA C | |
bADAMTS4 | Primer fw (5′–3′) | CCC CAT GTG CAA CGT CAA G |
Primer rev (5′–3′) | AGT CTC CAC AAA TCT GCT CAG TGA | |
Probe (5′FAM/3′TAMRA) | AGC CCC CGA AGG GCT AAG CGC | |
bADAMTS5 | Primer fw (5′–3′) | GAT GGT CAC GGT AAC TGT TTG CT |
Primer rev (5′–3′) | GCC GGG ACA CAC CGA GTA C | |
Probe (5′FAM/3′TAMRA) | AGG CCA GAC CTA CGA TGC CAG CC | |
bCA12 | Primer fw (5′–3′) | CTG CCA GTC CGC AAT TTG T |
Primer rev (5′–3′) | CCC CGG ACC TGC ATG TC | |
Probe (5′FAM/3′TAMRA) | CCA CTC AGT GAA GGT GAA ACT GCC CA | |
bKRT19 | Bt03219428_m1 | |
bFGFR1 | Bt03258913_m1 | |
bFGFR2 | Bt03649230_m1 | |
bFGFR3 | Bt03259318_m1 | |
bFGFR4 | Bt04292959_m1 | |
bRPLP0 | Bt03218086_m1 | |
hCOL1 | Primer fw (5′–3′) | CCC TGG AAA GAA TGG AGA TGA T |
Primer rev (5′–3′) | ACT GAA ACC TCT GTG TCC CTT CA | |
Probe (5′FAM/3′TAMRA) | CGG GCA ATC CTC GAG CAC CCT | |
hCOL2 | Primer fw (5′–3′) | GGC AAT AGC AGG TTC ACG TAC A |
Primer rev (5′–3′) | GAT AAC AGT CTT GCC CCA CTT ACC | |
Probe (5′FAM/3′TAMRA) | CCT GAA GGA TGG CTG CAC GAA ACA TAC | |
hACAN | Primer fw (5′–3′) | AGT CCT CAA GCC TCC TGT ACT CA |
Primer rev (5′–3′) | CGG GAA GTG GCG GTA ACA | |
Probe (5′FAM/3′TAMRA) | CCG GAA TGG AAA CGT GAA TCA GAA TCA ACT | |
hMMP3 | Hs00968305_m1 | |
hADAMTS4 | Hs00192708_m1 | |
hADAMTS5 | Hs01095518_m1 | |
hKRT19 | Hs00761767_s1 | |
hFGFR1 | Hs00241111_m1 | |
hFGFR2 | Hs01552918_m1 | |
hFGFR3 | Hs00179829_m1 | |
hFGFR4 | Hs01106910_g1 | |
hRPLP0 | Primer fw (5′–3′) | TGG GCA AGA ACA CCA TGA TG |
Primer rev (5′–3′) | CGG ATA TGA GGC AGC AGT TTC | |
Probe (5′FAM/3′TAMRA) | AGG GCA CCT GGA AAA CAA CCC AGC |
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Häckel, S.; Zolfaghar, M.; Du, J.; Hoppe, S.; Benneker, L.M.; Garstka, N.; Peroglio, M.; Alini, M.; Grad, S.; Yayon, A.; et al. Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells. Int. J. Mol. Sci. 2019, 20, 5036. https://doi.org/10.3390/ijms20205036
Häckel S, Zolfaghar M, Du J, Hoppe S, Benneker LM, Garstka N, Peroglio M, Alini M, Grad S, Yayon A, et al. Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells. International Journal of Molecular Sciences. 2019; 20(20):5036. https://doi.org/10.3390/ijms20205036
Chicago/Turabian StyleHäckel, Sonja, Mona Zolfaghar, Jie Du, Sven Hoppe, Lorin M. Benneker, Nathalie Garstka, Marianna Peroglio, Mauro Alini, Sibylle Grad, Avner Yayon, and et al. 2019. "Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells" International Journal of Molecular Sciences 20, no. 20: 5036. https://doi.org/10.3390/ijms20205036