Suppression of Hypertrophy During in vitro Chondrogenesis of Cocultures of Human Mesenchymal Stem Cells and Nasal Chondrocytes Correlates With Lack of in vivo Calcification and Vascular Invasion.

Matthew Anderson-Baron,Yan Liang,Aillette Mulet-Sierra,Khalid Ansari,Adetola B. Adesida,Melanie Kunze,Hadi Seikaly,Martin Osswald

doi:10.3389/fbioe.2020.572356

Abstract

ObjectiveHuman nasal septal chondrocytes (NC) are a promising minimally invasive derivable chondrogenic cell source for cartilage repair. However, the quality of NC-derived cartilage is variable between donors. Coculture of NC with mesenchymal stem cells (MSCs) mitigates the variability but with undesirable markers of chondrocyte hypertrophy, such as type X collagen, and the formation of unstable calcifying cartilage at ectopic sites. In contrast, monoculture NC forms non-calcifying stable cartilage. Formation of a stable NC-MSC coculture cartilage is crucial for clinical application. The aim of this study was to explore the utility of parathyroid hormone-related peptide (PTHrP) hormone to suppress chondrocyte hypertrophy in NC-MSC cocultures and form stable non-calcifying cartilage at ectopic sites.MethodsHuman NC and bone marrow MSCs, and cocultures of NC and MSC (1:3 ratio) were aggregated in pellet form and subjected to in vitro chondrogenesis for 3 weeks in chondrogenic medium in the presence and absence of PTHrP. Following in vitro chondrogenesis, the resulting pellets were implanted in immunodeficient athymic nude mice for 3 weeks.ResultsCoculture of NC and MSC resulted in synergistic cartilage matrix production. PTHrP suppressed the expression of hypertrophy marker, type X collagen (COL10A1), in a dose-dependent fashion without affecting the synergism in cartilage matrix synthesis, and in vivo calcification was eradicated with PTHrP. In contrast, cocultured control (CC) pellets without PTHrP treatment expressed COL10A1, calcified, and became vascularized in vivo.

Highlights

Advances in the field of tissue engineering have made tissueengineered cartilage a promising option for the treatment or replacement of damaged and diseased cartilage tissue
After 3 weeks of culture in chondrogenic media, pellets were harvested, the gross appearance of the pellets were captured on stereomicroscope (Figure 1A), the wet weight (WW) were recorded (Figure 1B) and in vitro chondrogenesis was first assessed by determining the GAG content relative to DNA content (Figure 1C)
Our findings offer the perspective that a small proportion of minimally expanded human Nasal chondrocytes (NC) from nasal septal cartilage can be used in conjunction with extensively expanded plastic-adherent cultured human bone marrow-derived mesenchymal stem cells (BM-MSCs) to generate stable implantable 3D engineered cartilage grafts

Summary

Introduction

Advances in the field of tissue engineering have made tissueengineered cartilage a promising option for the treatment or replacement of damaged and diseased cartilage tissue. Considerable challenges remain, including the acquisition of appropriate autologous sources of cells and the generation of stable engineered cartilage once implanted in vivo. NC have been shown to produce stable cartilage constructs in vivo and have been used to repair articular cartilage of the knee and reconstruct nasal cartilage in skin cancer patients (Fulco et al, 2014; Mumme et al, 2016); engineered cartilage from NC suffers from large donor-to-donor variability (Fulco et al, 2014; Andrews et al, 2017)

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Conclusion