Tgf β3

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TGF-β3 is a recombinant human transforming growth factor-beta 3 protein. It is a member of the transforming growth factor beta family and plays a role in various cellular processes.

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Recombinant human TGF-β3 (11 citations) Transforming Growth Factor β3 (TGF-β3) (4 citations)

181 protocols using tgf β3

Fabrication of Oriented ECM Scaffolds

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DC-ECM scaffolds were prepared by using temperature gradient-guided thermal-induced phase separation (TIPS) followed by freeze-drying (Figure 1).3 (link) Briefly, a plastic syringe with a flat bottom was surrounded by thermal insulation polystyrene material except for the top and bottom, and a mixed slurry with different composite of S/D at 3 wt% was loaded to form a mold-slurry device, which was placed onto the top of a cold liquid bath at −20°C to from a temperature gradient from the bottom to the top of the slurry. During the cold process, ice crystals in the slurry were formed and grew longitudinally from the bottom to the top. After 12 h, the frozen mold-slurry device was then quickly transferred into a lyophilizer and lyophilized for 24 h to obtain an oriented S/D composite scaffold. All scaffolds were cross-linked in an ethanol solution containing 50 mM 1-ethyl-3-3-dimethyl aminopropyl carbodiimide and 20 mM N-hydroxysuccinimide at 37°C for 24 h, then washed repeatedly with dH₂O. To develop the TGF-β3 delivery system, TGF-β3 was coated onto scaffolds saturated with a buffer solution containing TGF-β3 (Peprotech, Rocky Hill, NJ, USA; 400 ng/mL) at 4°C for 24 h. The TGF-β3-loaded scaffolds were then lyophilized. All processes were carried out under sterile conditions.

Yang Q., Teng B.H., Wang L.N., Li K., Xu C., Ma X.L., Zhang Y., Kong D.L., Wang L.Y, & Zhao Y.H. (2017). Silk fibroin/cartilage extracellular matrix scaffolds with sequential delivery of TGF-β3 for chondrogenic differentiation of adipose-derived stem cells. International Journal of Nanomedicine, 12, 6721-6733.

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Gelatin Scaffold Adsorption Protocols

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The gelatin scaffolds SPONGOSTAN® were obtained from Johnson & Johnson. Circular gelatin scaffolds were cut using a metal borer or punch with a diameter of 5 mm. They were sterilized by autoclave at 121°C, 15 pounds and 25 minutes. For the adsorption process, the scaffolds were transferred to a sterile 24-wells tissue culture plate and incubated with 100 μl solution of (a) 0.07% (w/v) HA (Viscoseal) in PBS (adsorb-HA), (b) 100 ng/ml TGF-β3 (Peprotech, USA) in PBS (adsorb-TGF-β3) or (c) 0.07% (w/v) HA and 100 ng/ml TGF-β3 in PBS (adsorb-HA + TGF-β3) and incubated at 37°C for 4 hours. A control scaffold was adsorbed with PBS alone in this step. For the soluble control group, the scaffold was processed the same as the PBS control group, and soluble 0.07% (w/v) HA and 10 ng/ml TGF-β3 were added directly into culture media each time the media was changed.

Klangjorhor J., Phitak T., Pruksakorn D., Pothacharoen P, & Kongtawelert P. (2014). Comparison of growth factor adsorbed scaffold and conventional scaffold with growth factor supplemented media for primary human articular chondrocyte 3D culture. BMC Biotechnology, 14, 108.

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Chondrogenic Differentiation of MenSCs and BMMSCs

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Both MenSCs and BMMSCs were detached, counted, and washed with DMEM medium without FBS by centrifugation at 500× g for 5 min. After the cells were transferred into the round bottom, low surface attachment 96 well plates (250,000 cells/well), centrifuged at 500× g for 5 min, and the medium was changed to chondrogenic medium, containing high glucose (4.5 g/L) DMEM, 1% PS (Merck), 1% insulin-transferrin-selenium (ITS) (Gibco Life Technologies, Waltham, MA, USA), 0.35 mM L-proline (Carl Roth, Karlsruhe, Germany), 10⁻⁷ M dexamethasone, 0.17 mM ascorbic acid phosphate (Sigma Aldrich) with or without growth factors. Growth factors—10 ng/mL TGF-β3 (Thermo Fisher Scientific), 50 ng/mL activin A, and 50 ng/mL BMP-2 and 100 ng/mL IGF-1—were added separately and as combinations of TGF-β3 + activin A, TGF-β3 + BMP-2, and TGF-β3 + IGF-1, using the same concentrations, where activin A, BMP-2, and IGF-1 were added only once at the beginning of differentiation. Chondrogenic differentiation was stimulated for 21 days and the chondrogenic medium was changed three times a week. During this time, cells formed pellets. After chondrogenic differentiation, the cell pellets were analyzed by RT-qPCR.

Uzieliene I., Bialaglovyte P., Miksiunas R., Lebedis I., Pachaleva J., Vaiciuleviciute R., Ramanaviciene A., Kvederas G, & Bernotiene E. (2023). Menstrual Blood-Derived Stem Cell Paracrine Factors Possess Stimulatory Effects on Chondrogenesis In Vitro and Diminish the Degradation of Articular Cartilage during Osteoarthritis. Bioengineering, 10(9), 1001.

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Chondrocyte Differentiation Protocol

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Primary chondrocytes were cultured in the customized SFM (CM) and maintained for 6 days. The CM was composed of DMEM/F12 (Gibco), 2% B27 (Gibco), 30 ng mL⁻¹ bFGF (PeproTech), and 10 ng mL⁻¹ TGFβ3 (PeproTech). The medium was refreshed every 3 days during stage 1. After 6 days, the medium was changed to a chondrogenic differentiation medium for another 21 days. The medium was composed of H‐DMEM (Gibco), 10 ng mL⁻¹ TGFβ3 (PeproTech), 10⁻⁷m dexamethasone (Sigma), 1% ITS (Gibco), 50 mg mL⁻¹ ascorbic acid (Sigma), and 1 mm sodium pyruvate (Gibco). This medium was also changed every 3 days during stage 2. As a control, chondrocytes were cultured in a typical serum‐containing medium (TM) which was composed of DMEM/F12 (Gibco) and 10% FBS (Gibco) for 6 days. Then, the medium was changed to a chondrogenic differentiation medium for another 21 days. The frequency of medium refresh was synchronized with the CC group.

Wen Y., Chen Y., Wu W., Zhang H., Peng Z., Yao X., Zhang X., Jiang W., Liao Y., Xie Y., Shen X., Sun H., Hu J., Liu H., Chen X., Chen J, & Ouyang H. (2023). Hyperplastic Human Macromass Cartilage for Joint Regeneration. Advanced Science, 10(26), 2301833.

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Fabrication of PLGA-CECM Composite Scaffolds

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Freeze-dried CECM was resuspended in deionizer water to form a 3% (w/v) suspension. An adequate amount of KGN (MedChemexpress, Rocky Hill, NJ, United States) was then added to the suspension. The mixture was poured into a cylindrical mould, frozen at −80°C for 72 h, and freeze-dried for 48 h. The resulting scaffolds were sterilized and trimmed to an appropriate size. Next, cross-linking was performed in anhydrous ethanol solution containing 50 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and 20 mM N-hydroxysuccinimide (NHS) for 24 h at 4°C; cross-linked scaffolds were rinsed several times with PBS. The process produced PLGA microsphere-embedded CECM composite scaffolds with diameter of approximately 5 mm and depth of approximately 3 mm. Scaffolds loaded with TGF-β3 were obtained by immersion in 400 ng/mL TGF-β3 (Peprotech, Rocky Hill, NJ, United States) solution (24 h at 4°C) and then freeze-dried.

Zhao Y., Zhao X., Zhang R., Huang Y., Li Y., Shan M., Zhong X., Xing Y., Wang M., Zhang Y, & Zhao Y. (2020). Cartilage Extracellular Matrix Scaffold With Kartogenin-Encapsulated PLGA Microspheres for Cartilage Regeneration. Frontiers in Bioengineering and Biotechnology, 8, 600103.

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Hypoxic Chondrocyte Differentiation of hESCs

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HUES7 hESCs (Howard Hughes Medical Institute/Harvard University, USA) were cultured at 5% O₂ (hypoxia) on Matrigel coated plates as described previously^{40 (link)}. Cells were passaged at ~ 80% confluency using Collagenase IV. hESCs were differentiated into chondrocytes at either 20% O₂, or 5% O₂ in medium (DMEM: F12 containing 1 × non-essential amino acids, 1 × B27 supplement, 90 μM β-mercaptoethanol and 1X ITS supplement: 10 μg/ml insulin, 5.5 μg/ml transferrin and 5 ng/ml selenite premix) supplemented with growth factors over a 14-day period. Medium was replenished daily. Growth factors were added as follows: Day 1, 25 ng/ml WNT3A (R&D) and 50 ng/ml Activin-A (Peprotech); Day 2, 25 ng/ml WNT3A, 25 ng/ml Activin-A and 20 ng/ml FGF2 (Invitrogen); Day 3, 25 ng/ml WNT3A, 10 ng/ml Activin-A, 20 ng/ml FGF2 and 40 ng/ml BMP4 (Peprotech); Days 4–7, 20 ng/ml FGF2, 40 ng/ml BMP4, 100 ng/ml Follistatin (Sigma) and 2 ng/ml NT4 (Peprotech); Day 8, 20 ng/ml FGF2, 40 ng/ml BMP4 and 2 ng/ml NT4; Day 9 and 10, 20 ng/ml FGF2, 20 ng/ml BMP4, 20 ng/ml GDF5 (Peprotech), 2 ng/ml NT4 and 10 ng/ml TGF-β3 (Peprotech); Days 11–14, 20 ng/ml FGF2, 40 ng/ml GDF5, 2 ng/ml NT4 and 10 ng/ml TGF-β3. Cells were dissociated using collagenase IV and passaged at a ratio of 1:2 or 1:3 on days 4 and 9 of the protocol.

Griffith L.A., Arnold K.M., Sengers B.G., Tare R.S, & Houghton F.D. (2021). A scaffold-free approach to cartilage tissue generation using human embryonic stem cells. Scientific Reports, 11, 18921.

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EMT and MET induction in NMuMG cells

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NMuMG; non-tumorigenic mouse mammary gland cell line, Hepa1-6; mouse hepatoma cell line, CMT; mouse polyploid carcinoma cell line and HEK-293; human embryonic kidney cell line were obtained from ATCC. Cells were maintained in DMEM (Gibco) and 10% fetal bovine serum (FBS) at 37 °C, 10% CO 2 . NMuMG medium was supplemented with 10 μg/ml insulin (Sigma). EMT induction of NMuMG cells was done by applying 5 ng/ml TGFβ3 (PeproTech) for 72 h. The reversible transition to the epithelial state (MET) was induced by TGFβ3 withdrawal, washing the plates twice with PBS and incubation for additional 72 h in fresh medium.

Alotaibi H., Basilicata M.F., Shehwana H., Kosowan T., Schreck I., Braeutigam C., Konu O., Brabletz T, & Stemmler M.P. (2015). Enhancer cooperativity as a novel mechanism underlying the transcriptional regulation of E-cadherin during mesenchymal to epithelial transition. Biochimica et biophysica acta, 1849(6).

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Cell-Laden Alginate Hydrogel Scaffold

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In this study, RGD-coupled alginate (NovaMatrix FMC Biopolymer, Norway) was used as the scaffold material. The alginate was purified and partially oxidized (2%) to increase its degradability according to published methods in the literature [28 (link)–30 (link)]. Subsequently, the alginate was concentrated, freeze-dried under reduced pressure, and mixed with TGF-β3 (Invitrogen, Carlsbad, CA) (50 μg/mL).
PDLSCs, GMSCs, and hBMMSCs (as a positive control) were encapsulated separately in alginate loaded with TGF-β3 ligand. Cells were encapsulated at a density of 2×10⁶ cells/mL of alginate solution. Microsphere formation was accomplished by adding the MSC-alginate mixture dropwise to 100 mM CaCl₂ solution. The resulting microspheres were incubated at 37°C for 45 min to complete cross-linking and then washed three times in non-supplemented DMEM. RGD-coupled alginate hydrogel without cells (and not loaded with TGF- β3) was used as the negative control in this study.

Moshaverinia A., Xu X., Chen C., Ansari S., Zadeh H.H., Snead M.L, & Shi S. (2014). Application of stem cells derived from the periodontal ligament or gingival tissue sources for tendon tissue regeneration. Biomaterials, 35(9), 2642-2650.

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Chondrogenic Differentiation of MSCs

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Passage 4 MSCs at 90% confluency were trypsinized and pelleted. To make MSC pellet culture, cells were resuspended in full chondrogenic medium (CM: high-glucose Dulbecco’s modified Eagle medium supplemented with 1% antibiotics-antimycotics, 0.1 μM dexamethasone (Sigma), 50 μg/mL ascorbate 2-phosphate (Sigma), 40 μg/mL L-proline (Sigma), 10 μg/mL ITS+ (Thermo Fisher, Waltham, MA), 10 ng/mL TGFβ3 (Peprotech, Rocky Hill, NJ)) at a concentration of 1 × 10⁶ cells/mL. Aliquots (200 μL) containing 2 × 10⁵ MSCs were pipetted into individual wells of conical-bottom 96-well plates (Thermo Fisher) and pelleted by centrifugation (300g, 10 min).

Deng Y., Lei G., Lin Z., Yang Y., Lin H, & Tuan R.S. (2018). Engineering Hyaline Cartilage from Mesenchymal Stem Cells with Low Hypertrophy Potential via Modulation of Culture Conditions and Wnt/β-Catenin Pathway. Biomaterials, 192, 569-578.

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Chondrogenic Micromass Culture Protocol

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A total of 2.5 × 10⁵ cells (P2) per micromass were suspended in 10-20 μL basal medium, pipetted carefully into a six-well plate and incubated for 1 h allowing attachment to the plate and cell/cell adhesion. Basal medium was removed and chondrogenic medium (α-MEM, 10 nM Dex, 100 μM Asc, 10 ng/mL TGFβ-3 [Peprotech, London, W6 8LL, United Kingdom]) and 10 μg/mL insulin (in the form of 100× insulin/tranferrin/sodium selenite [ITS] solution) was carefully added so as to not disturb the micromass. After 21 days, cultures were fixed in 4 % PFA and stained with Alcian blue (0.5 % w/v in acidic H₂O [0.01 % acetic acid]).

Gothard D., Cheung K., Kanczler J.M., Wilson D.I, & Oreffo R.O. (2015). Regionally-derived cell populations and skeletal stem cells from human foetal femora exhibit specific osteochondral and multi-lineage differentiation capacity in vitro and ex vivo. Stem Cell Research & Therapy, 6, 251.

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