recombi ant human gdf5  (R&D Systems)

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 1. SDS-PAGE and densitometric analy- ses of cell layers of human tenocytes treated without ( − )/with ( + ) MMC (carrageenan) and without ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that MMC supple- mentation, at all timepoints, induced signifi- cantly ( p < 0.05) higher collagen type I depo- sition than the non-MMC groups (without or even with any GF). among the simultaneous GF supplementation to MMC, TGF 𝛽3 induced the highest ( p < 0.05) collagen type I deposition in human tenocyte cultures at all time points. among the serial GF supplementation to MMC, TGF 𝛽3 induced the highest ( p < 0.05) colla- gen type I deposition in human tenocyte cul- tures at all time points. Day 4 ( A ), Day 7 ( B ), Day 10 ( C ), Day 13 ( D ). ∗ indicates significantly ( p < 0.05) higher difference between the simul- taneous and/or serial GF supplementation to MMC and MMC alone groups. Passage 3. N = 3.

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: SDS Page

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 2. Immunocytochemistry and relative flu- orescence intensity analyses of collagen type I of cell layers of human tenocytes treated with- out ( − )/with ( + ) MMC (carrageenan) and with- out ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that MMC supplementa- tion, at all timepoints, induced significantly ( p < 0.05) higher collagen type I deposition than the non-MMC groups (without or even with any GF). At day 10 and 13, IGF1 in both simultaneous and serial to MMC supplementa- tion induced significantly higher ( p < 0.05) col- lagen type I deposition than the MMC alone group ( A ). No significant ( p > 0.05) difference was detected in collagen type I deposition be- tween MMC alone and PDGF 𝛽𝛽supplementa- tion in either simultaneous or serial fashion to MMC ( B ). At days 4, 10 and 13, GDF5 sup- plementation in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type I deposition than the MMC alone group ( C ). At day 4, 7 and 13, TGF 𝛽3 supplementation in si- multaneous and serial fashion to MMC induced significantly higher ( p < 0.05) collagen type I deposition than the MMC alone group ( D ). ∗

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: Immunocytochemistry

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 3. Immunocytochemistry and relative flu- orescence intensity analyses of collagen type III of cell layers of human tenocytes treated with- out ( − )/with ( + ) MMC (carrageenan) and with- out ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that MMC supplementa- tion, at all timepoints, induced significantly ( p < 0.05) higher collagen type III deposition than the non-MMC groups (without or even with any GF). At day 7 and, IGF1 supplementa- tion in simultaneous and serial fashion to MMC induced significantly higher ( p < 0.05) collagen type III deposition than the MMC alone group ( A ). No significant ( p > 0.05) difference was de- tected in collagen type III deposition between MMC alone and PDGF 𝛽𝛽supplementation in either simultaneous or serial fashion to MMC ( B ). At day 10 and 13, GDF5 supplementation in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type III deposition than the MMC alone group ( C ). At day 4 and 7, TGF 𝛽3 supplementation either simultaneously or in serial fashion to MMC and at day 13 only in serial fashion to MMC induced significantly ( p < 0.05) higher collagen type III deposition than the MMC alone group ( D ). ∗ indicates sig- nificantly (at p < 0.05) higher difference be- tween the simultaneous and/or serial GF sup- plementation to MMC and MMC alone groups. Collagen type III: Green. DAPI: Blue. Scale bars: 100 𝜇m. Passage 3. N = 3.

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: Immunocytochemistry

Journal: Biomaterials and Biosystems

Article Title: Growth factor and macromolecular crowding supplementation in human tenocyte culture

doi: 10.1016/j.bbiosy.2021.100009

Figure Lengend Snippet: Fig. 4. Hierarchal clustering of the fold change (threshold of 3) in gene expression of human tenocytes cultured without ( − )/with ( + ) MMC (carrageenan) and without ( − )/with ( + ) (either in simultaneous or in serial fashion to MMC) IGF1, PDGF 𝛽𝛽, GDF5 and TGF 𝛽3 revealed that at all timepoints TGF 𝛽3 alone and TGF 𝛽3 in either simultaneous or serial supplementation to CR upregulated the most and downregulated the least collagen- and tendon- related genes and upregulated the least and downregulated the most osteo-, chondro-, fibrosis- and adipose- related trans-differentiation genes in comparison to the other GFs without CR and with CR (either in simultaneous or serial fashion to the GFs). IGF1 ( A ), PDGF 𝛽𝛽( B ), GDF5 ( C ), TGF 𝛽3 ( D ). The heatmap was generated by a log transformation of the real-time PCR data presented as ΔCT = (CT miRNA – CT GAPDH) compared to without CR and without GF at each timepoint. Passage 3. Data derived by pooling six wells per sample ( N = 2).

Article Snippet: GF and MMC supplementation At passage three, tenocytes were seeded at 25,000 cells/cm 2 in 24- ell plates and allowed to attach for 24 h. The culture media was re- oved and replaced with culture media containing 100 μM L-ascorbic cid phosphate (Sigma Aldrich, Ireland) to induce collagen synthesis nd 100 ng/ml recombinant human IGF1 (R&D Systems, UK), 50 ng/ml ecombinant human PDGF ββ (PeproTech EC, UK), 100 ng/ml recombi- ant human GDF5 (R&D Systems, UK) or 20 ng/ml recombinant human GF β3 (PeproTech EC, UK) with and without 50 μg/ml of carrageenan CR, mixture of κ and lesser amounts of λ CR, C1013, Sigma Aldrich, reland).

Techniques: Gene Expression, Cell Culture, Comparison, Generated, Transformation Assay, Real-time Polymerase Chain Reaction, Derivative Assay