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Glucosamine Hydrochloride and N-Acetylglucosamine Influence the Response of Bovine Chondrocytes to TGF-β3 and IGF in Monolayer and Three-Dimensional Tissue Culture

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Tissue Engineering and Regenerative Medicine Aims and scope

Abstract

Background:

Glucosamine hydrochloride (GlcN·HCl) has been shown to inhibit cell growth and matrix synthesis, but not with N-acetyl-glucosamine (GlcNAc) supplementation. This effect might be related to an inhibition of critical growth factors (GF), or to a different metabolization of the two glucosamine derivatives. The aim of the present study was to evaluate the synergy between GlcN·HCl, GlcNAc, and GF on proliferation and cartilage matrix synthesis.

Method:

Bovine chondrocytes were cultivated in monolayers for 48 h and in three-dimensional (3D) chitosan scaffolds for 30 days in perfusion bioreactors. Serum-free (SF) medium was supplemented with either growth factors (GF) TGF-β (5 ng mL−1) and IGF-I (10 ng mL−1), GlcN·HCl or GlcNAc at 1mM each or both. Six groups were compared according to medium supplementation: (a) SF control; (b) SF + GlcN·HCl; (c) SF + GlcNAc; (d) SF + GF; (e) SF + GF + GlcN·HCl; and (f) SF + GF + GlcNAc. Cell proliferation, proteoglycan, collagen I (COL1), and collagen II (COL2) synthesis were evaluated.

Results:

The two glucosamines showed opposite effects in monolayer culture: GlcN·HCl significantly reduced proliferation and GlcNAc significantly augmented cellular metabolism. In the 30 days 3D culture, the GlcN·HCl added to GF stimulated cell proliferation more than when compared to GF only, but the proteoglycan synthesis was smaller than GF. However, GlcNAc added to GF improved the cell proliferation and proteoglycan synthesis more than when compared to GF and GF/GlcN·HCl. The synthesis of COL1 and COL2 was observed in all groups containing GF.

Conclusion:

GlcN·HCl and GlcNAc increased cell growth and stimulated COL2 synthesis in long-time 3D culture. However, only GlcNAc added to GF improved proteoglycan synthesis.

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Acknowledgement

This study was supported by grants from CAPES Foundation within the Ministry of Education of Brazil, University of Bayreuth, and the Friedrich-Baur Biomed Center.

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Authors and Affiliations

  1. Laboratory of Biomechanical Engineering (LEBm), University Hospital, Department of Mechanical Engineering, Federal University of Santa Catarina, St. Maria Flora Pausewang, Florianópolis, SC, 88036-800, Brazil

    André Luiz A. Pizzolatti, Carlos R. M. Roesler & Gean Vitor Salmoria

  2. CAPES Foundation, Ministry of Education of Brazil, St. ERL-Norte, Brasília, DF, 70.040-020, Brazil

    André Luiz A. Pizzolatti

  3. Friedrich Baur Biomed Center, Bayreuth, St. Ludwig-Thoma- 36c, 95447, Bayreuth, Bavaria, Germany

    Florian Gaudig & Daniel Seitz

  4. University of Bayreuth, St. University 30, 95447, Bayreuth, Bavaria, Germany

    Florian Gaudig & Daniel Seitz

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  1. André Luiz A. Pizzolatti
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Supplementary material 1.

The effect of GlcN·HCl and GlcNAc doses on cell proliferation and viability. A dsDNA quantification (F statistic p < 0.001). * = p < 0.05, compared to 0.5 mM. ** = p < 0.001, compared to 1 mM and 10 mM. # = p < 0.05, compared to 0 mM. B Effect of GlcN·HCl (F statistic, p = 0.24) and GlcNAc (F statistic, p = 0.69) on NADPH/NADH2 synthesis. C Lactate synthesis, (F statistic p < 0.001). * = p < 0.01 compared to 0, 1 µM, and 0.5 mM. # = p < 0.05 compared to 5 mM. § = p < 0.05 compared to control, 1 µM, and 0.5 mM. ƚ = p < 0.001 compared to 5 mM and 10 mM. (PPTX 57 kb)

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Pizzolatti, A.L.A., Gaudig, F., Seitz, D. et al. Glucosamine Hydrochloride and N-Acetylglucosamine Influence the Response of Bovine Chondrocytes to TGF-β3 and IGF in Monolayer and Three-Dimensional Tissue Culture. Tissue Eng Regen Med 15, 781–791 (2018). https://doi.org/10.1007/s13770-018-0150-x

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