Int J Stem Cells.  2023 Aug;16(3):304-314. 10.15283/ijsc21242.

Hypoxia Differentially Affects Chondrogenic Differentiation of Progenitor Cells from Different Origins

• Hammad M
• Veyssiere A
• Leclercq S
• Patron V
• Baugé C
• Boumédiene K

• KMID: 2545230
• DOI: http://doi.org/10.15283/ijsc21242

Abstract

Background and Objectives
Ear cartilage malformations are commonly encountered problems in reconstructive surgery, since cartilage has low self-regenerating capacity. Malformations that impose psychological and social burden on one’s life are currently treated using ear prosthesis, synthetic implants or autologous flaps from rib cartilage. These approaches are challenging because not only they request high surgical expertise, but also they lack flexibility and induce severe donor-site morbidity. Through the last decade, tissue engineering gained attention where it aims at regenerating human tissues or organs in order to restore normal functions. This technique consists of three main elements, cells, growth factors, and above all, a scaffold that supports cells and guides their behavior. Several studies have investigated different scaffolds prepared from both synthetic or natural materials and their effects on cellular differentiation and behavior.
Methods and Results
In this study, we investigated a natural scaffold (alginate) as tridimensional hydrogel seeded with progenitors from different origins such as bone marrow, perichondrium and dental pulp. In contact with the scaffold, these cells remained viable and were able to differentiate into chondrocytes when cultured in vitro. Quantitative and qualitative results show the presence of different chondrogenic markers as well as elastic ones for the purpose of ear cartilage, upon different culture conditions.
Conclusions
We confirmed that auricular perichondrial cells outperform other cells to produce chondrogenic tissue in normal oxygen levels and we report for the first time the effect of hypoxia on these cells. Our results provide updates for cartilage engineering for future clinical applications.

Keyword

Chondrogenesis; Stem cells; Cartilage engineering; Hypoxia

Figure

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  Fig. 1 Multilineage differentiation potential of human progenitors. The trilineage differentiation of AuP, DPSC and NsP were carried out by Alizarin red (a∼c), oil red O (d∼f), and alcian blue (g∼i), respectively. (j∼l): Bright field images of the cells. These results were compared to a control (Supplementary Fig. S1). Scale 500 μm.

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  Fig. 2 Production of GAGs and glycoproteins after 2 weeks of culture. Safranin O and alcian blue staining were respectively performed for illustrating the positive production of proteoglycans (red) and glycosaminogly-cans (blue) in bone marrow-derived mesenchymal stem cells (BMMSC) a, e, i, m, auricular perichondrocytes (AuP) b, f, j, n dental stem cells (DPSC) c, g, k, o, and nasal perichondrocytes (NsP) d, h, l, p, encapsulated in 3D alginate microsphere in normoxic and hypoxic conditions, respectively, after 2 weeks of culture in chondrogenic medium (scale bar 500 μm).

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  Fig. 3 Expression of cartilage genes in four different cell sources in Normoxic and hypoxic environments. RT-PCR analysis were performed to examine cartilaginous markers in 4 different cell lines in normoxic and hypoxic conditions. The expression of these markers was compared to the average of the 3 housekeeping genes. *p<0.05, **p<0.01, ***p<0.005.

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  Fig. 4 Immunohistochemistry of ELN, COL II and ACAN in presence and absence of oxygen tension. (A) Negative control was illustrated where primary antibody was replaced by PBS (a∼c). (B∼D) Type 2 collagen, aggrecan and elastin expression was detected in auricular perichondrocytes and bone marrow-derived mesenchymal stem cells in presence of high (a∼c, g∼i) and low oxygen tension (d∼f, j∼l), respectively (scale bar 200 μm).

Reference

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