Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: The amino acid substitutions of GDF5 variants A and B.

Article Snippet: Therefore, we used four different recombinant GDF5 proteins in total: wildtype mouse (R&D Systems), wildtype human (Biopharm) and the two variants, A and B.

Techniques: Variant Assay

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Y-axis represents the luciferase activity readings generated in SW1353 cells in response to exogenous growth factor. Cells were stimulated for 6, 12, 24 and 48 hours with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C) and human GDF5 variant B (D). BMP2 and TGF-β1 stimulations were used as positive controls. Error bars represent the standard error of the mean. GDF5 10, 10 ng/ml; GDF 30, 30 ng/ml; GDF5 100, 100 ng/ml; GDF5 300, 300 ng/ml. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, *****P<0.00001, two-tailed Student’s t-test.

Article Snippet: Therefore, we used four different recombinant GDF5 proteins in total: wildtype mouse (R&D Systems), wildtype human (Biopharm) and the two variants, A and B.

Techniques: Luciferase, Activity Assay, Generated, Variant Assay, Two Tailed Test

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were cultured in monolayer and stimulated with each of the four GDF5 proteins for 15 minutes, 30 minutes, 1-β1 stimulation for 1 hour was used as a positive control. Protein was extracted and subjected to western blot analysis using an antibody against Smad 1/5/8 (anti-Smad1/5/8), with anti-β-actin antibody used as a loading control. This data comes from one OA patient. Identical data was obtained for a second OA patient (data not shown).

Article Snippet: Therefore, we used four different recombinant GDF5 proteins in total: wildtype mouse (R&D Systems), wildtype human (Biopharm) and the two variants, A and B.

Techniques: Cell Culture, Positive Control, Western Blot, Control

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were stimulated for 6, 12, 24 and 48 hours with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C), human GDF5 variant B (D) and TGF-β1 (E). Each cross represents a significant (P<0.05, two-tailed Student’s t-test) up/down regulation of gene expression relative to untreated cells in one patient. Twelve patients were studied for each of the GDF5 growth factor treatments and ten patients for the TGF-β1 treatments.

Article Snippet: Therefore, we used four different recombinant GDF5 proteins in total: wildtype mouse (R&D Systems), wildtype human (Biopharm) and the two variants, A and B.

Techniques: Variant Assay, Two Tailed Test, Gene Expression

Journal: PLoS ONE

Article Title: Human Chondrocytes Respond Discordantly to the Protein Encoded by the Osteoarthritis Susceptibility Gene GDF5

doi: 10.1371/journal.pone.0086590

Figure Lengend Snippet: Chondrocytes were stimulated for 5 days with wildtype mouse GDF5 (A), wildtype human GDF5 (B), human GDF5 variant A (C), human GDF5 variant B (D) and TGF-β1 (E). Each cross represents a significant (P<0.05, two-tailed Student’s t-test) up/down regulation of gene expression relative to untreated cells in one patient. Ten patients were studied for each of the growth factor treatments.

Article Snippet: Therefore, we used four different recombinant GDF5 proteins in total: wildtype mouse (R&D Systems), wildtype human (Biopharm) and the two variants, A and B.

Techniques: Variant Assay, Two Tailed Test, Gene Expression

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: In vitro release of 2 and 10 µg/mL GDF5 and BB-1 from CPC. Cement discs ( r = 2.0 mm, h = 2.0 mm, V = 25.12 µL) were loaded with 2 or 10 µg/mL GDF5 and BB-1, ground, and incubated at 37 °C in 1 mL of cell culture medium + 10% FCS for 1 h, as well as 1, 2, 3, 6, 8, 10, 13, 15, 17, 20, 22, 28, and 31 days. The release of GDF5 ( A ) and BB-1 ( E ) from ground cement discs was measured by ELISA and used for the calculation of cumulative release ( B , F ), % release ( C , G ), and retention ( D , H ; n = 2).

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: In Vitro, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: In vitro release of 200 and 1000 µg/mL GDF5 and BB-1 from CPC. Cement cuboids ( l = 10 mm, h = 5.0 mm, w = 5.0 mm, V = 250 µL) were loaded with 200 or 1000 µg/mL GDF5 and BB-1, ground, and incubated at 37 °C in 2 mL of either PBS or sheep serum. After 1 h, as well as 1, 2, 5, 7, 9, 12, 14, 16, 19, 22, 27, and 30 days, the release of GDF5 ( A ) and BB-1 ( E ) from ground cement discs was measured by ELISA and used for the calculation of cumulative release ( B , F ), % release ( C , G ), and retention ( D , H ; n = 1).

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: In Vitro, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Release of GDF5, BB-1, and BMP-2 from PLGA fiber-reinforced CPC. Discs consisting of PLGA fiber-reinforced CPC ( r = 4.0 mm, h = 0.5 mm, V = 25.12 µL) were loaded with 400 µg/mL GDF5, BB-1, and BMP-2, ground, and incubated at 37 °C in 1 mL of sheep serum. After 1 h, as well as 1, 3, 7, and 14 days, the release of GDF5 ( A ), BB-1 ( E ), and BMP-2 ( I ) from ground cement discs was measured by ELISA and used for the calculation of cumulative release ( B , F , J ), % release ( C , G , K ), and retention ( D , H , L ). Data are expressed as means ± SEM ( n = 3). * p ≤ 0.05 Mann–Whitney U test vs. pure CPC. + p ≤ 0.05 Mann–Whitney U test vs. 5% fibers.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Differences among the release of the different BMPs from ground pure CPC and CPC ± PLGA fibers (GDF5, BB-1, BMP-2; n = 3 experiments each); # p ≤ 0.05 vs. GDF5; § p ≤ 0.05 vs. BB-1; note: differences among the different release time points are only shown in Figure 3 .

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques:

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Alkaline phosphatase (ALP) activity in C2C12 cells. C2C12 cells were cultivated for 3 days in diluted extracts of GDF5, BB-1, or BMP-2-containing, ground pure CPC or PLGA fiber-reinforced CPC. Thereafter, the ALP activity was measured using an ALP assay and the data were expressed as fold-change induction compared to the CPC without growth factor; data are expressed as means ± SEM ( n = 3); n.d. = not determined; a.u. = arbitrary units.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Activity Assay, ALP Assay

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Alkaline phosphatase (ALP) activity in C2C12BRIb cells. C2C12BRIb cells were cultivated for 3 days in diluted extracts of GDF5, BB-1, or BMP-2-containing, ground pure CPC or PLGA fiber-reinforced CPC. Thereafter, the ALP activity was measured using an ALP assay and the data were expressed as fold-change induction compared to the CPC without growth factor; data are expressed as means ± SEM ( n = 3); n.d. = not determined; a.u. = arbitrary units.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Activity Assay, ALP Assay

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Alkaline phosphatase (ALP) activity in MCHT-1/26 cells. MCHT-1/26 cells were cultivated for 3 days in diluted extracts of GDF5, BB-1, or BMP-2-containing, ground pure CPC or PLGA fiber-reinforced CPC. Thereafter, the ALP activity was measured using an ALP assay and the data were expressed as fold-change induction compared to the CPC without growth factor; data are expressed as means ± SEM ( n = 3); n.d. = not determined; a.u. = arbitrary units.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Activity Assay, ALP Assay

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Alkaline phosphatase (ALP) activity in ATDC5 cells. ATDC5 cells were cultivated for 3 days in diluted extracts of GDF5, BB-1, or BMP-2-containing, ground pure CPC or PLGA fiber-reinforced CPC. Thereafter, the ALP activity was measured using an ALP assay and the data were expressed as fold-change induction compared to the CPC without growth factor; data are expressed as means ± SEM ( n = 3); n.d. = not determined; a.u. = arbitrary units.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Activity Assay, ALP Assay

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Effects of GDF5 extracts on the gene expression in hASCs. hASCs were seeded in 12-well plates and exposed for 3 days to the diluted extracts of CPC discs without fibers (CPC) and with 10% ( w / w ) fiber content (CPC+F), the latter either without GDF5 or doped with low dose (10 µg/mL; CPC+F+G) or high dose GDF5 (200 µg/mL; CPC+F+hG). Thereafter, the mRNA expression of Runx2 (RUNX; A ), osterix ( B ), alkaline phosphatase (ALP; C ), type I collagen ( D ), osteopontin ( E ), osteocalcin ( F ), type II collagen ( G ), and aggrecan ( H ) was measured using RT-PCR and the data were expressed as relative expression (as determined using the ΔΔCt method) compared to the CPC without fibers and growth factor; data are expressed as means ± SEM ( n = 8); * p ≤ 0.05 Mann–Whitney U test vs. CPC; # p ≤ 0.05 Mann–Whitney U test vs. CPC+F.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Gene Expression, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY

Journal: Pharmaceutics

Article Title: In Vitro Release of Bioactive Bone Morphogenetic Proteins (GDF5, BB-1, and BMP-2) from a PLGA Fiber-Reinforced, Brushite-Forming Calcium Phosphate Cement

doi: 10.3390/pharmaceutics11090455

Figure Lengend Snippet: Effects of GDF5 extracts on the intracellular concentration of collagen 1 protein in hASCs. hASCs were seeded in 12-well plates and exposed for 3 days to the diluted extracts of CPC discs without fibers (CPC) and with 10% ( w / w ) fiber content (CPC+F), the latter either without GDF5 or doped with low dose (10 µg/mL; CPC+F+G) or high dose GDF5 (200 µg/mL; CPC+F+hG). Thereafter, collagen 1 concentrations in cell lysates were quantified using a commercial ELISA kit. The data are expressed as means ± SEM ( n = 8); # p ≤ 0.05 Mann–Whitney U test vs. CPC+F.

Article Snippet: The recombinant human (rh) growth factors GDF5 (also called BMP-14), BB-1, and BMP-2 were produced in E. coli by the company BIOPHARM GmbH (Heidelberg, Germany; GDF5, BB-1) and the Hans-Knöll-Institut in Jena (BMP-2) using patented procedures [ ].

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY