Elsevier

Acta Histochemica

Volume 122, Issue 6, September 2020, 151588
Acta Histochemica

Characterization of human articular chondrocytes and chondroprogenitors derived from non-diseased and osteoarthritic knee joints to assess superiority for cell-based therapy

https://doi.org/10.1016/j.acthis.2020.151588Get rights and content

Highlights

  • Cultured chondrocytes and chondroprogenitors exhibit similar characteristics.

  • Both demonstrate multilineage potential.

  • Chondroprogenitors show higher GAG and lower calcified matrix deposition.

  • Sorting based on CD34- and CD166+ will provide chondroprogenitors of high purity.

Abstract

Purpose

Cell based therapy is constantly underway since regeneration of genuine hyaline cartilage is under par. Much attention has been afforded to chondroprogenitors recently, as an alternative cell substitute for cartilage repair. Although single source derivation of chondrocytes and chondroprogenitors is advantageous, lack of a characteristic differentiating marker obscures clear identification, which is essential to create a biological profile and is also required to assess cell type superiority for cartilage repair.

Methods

Cells obtained from three non-diseased/osteoarthritic human knee joints each, were expanded in culture up to passage 10. Characterization studies were performed using flow cytometry; gene expression was studied using RT-PCR; growth kinetics and tri-lineage differentiation was also studied to construct a better profile of chondroprogenitors as well as chondrocytes.

Results and conclusion

Our results showed that both cell populations exhibited similar cell surface characteristics except for non-diseased chondroprogenitors, which showed markedly low expression of CD34 and high expression of CD166. Trilineage data was suggestive of multilineage potential for both cell types with chondroprogenitors showing notably higher glycosaminoglycan and lower calcified matrix deposition. Data acquired from this study aided in describing cellular behavior of human articular cartilage derived chondroprogenitors in conditions not reported earlier. Our comparative analysis suggests that sorting based on a combination of markers (CD34- and CD166+) would yield a population of cells with minimal contamination by chondrocytes, which may provide translatable results in terms of enhanced chondrogenesis and reduced hypertrophy; both indispensable for the field of cartilage regeneration.

Introduction

Much focus has been directed to using cell-based therapy in treatment of cartilage afflictions like osteoarthritis (OA) and osteo-chondral defects (Dall’Oca et al., 2017). The main contenders in this field which have been used as either stand-alone substitutes, as co-cultures or in combination with scaffolds and growth factors are cartilage derived chondrocytes and mesenchymal stem cells(MSCs) (Grässel and Anders, 2012; Mobasheri et al., 2014). Although MSCs (due to inherent multipotency and high replicative potential) and chondrocytes (tissue nativity making them safe for use) are promising candidates; their current usage still warrants optimization (Akkiraju and Nohe, 2015; Oreffo et al., 2005). It has been reported that MSCs exhibit a tendency for increased osteogenesis in-vitro and fibrocartilage formation in vivo (Akgun et al., 2015; Fernandez-Moure et al., 2015; Freitag et al., 2016; Liu et al., 2017). Similarly, the limitations that affect chondrocyte use (Autologous chondrocyte implantation) are graft hypertrophy and mixed fibro-hyaline formation (Harris et al., 2011; Pareek et al., 2016). Moreover, chondrocytes require expansion in- vitro, since cell yield post-harvest is too low to meet demands of direct implantation. This raises another conflict since there does not appear to be a consensus on chondrocyte behavior in culture. There is evidence to show that with increased time in culture, chondrocytes lose their phenotype and show higher expression of markers for hypertrophy thereby reducing their efficiency for optimal cartilage repair (Ma et al., 2013). However, there are also reports which demonstrate that chondrocytes exhibit positive stem cell markers in culture (Diaz-Romero et al., 2008; Hamada et al., 2013; Vinod et al., 2019)

Continued search for an optimal cell source led to a potential cell type residing within the superficial layer of cartilage (Dowthwaite et al., 2004). Isolated by fibronectin adhesion assay, articular cartilage derived chondroprogenitors have been classified as MSCs since they demonstrate similar marker profile (Notch-1 signaling proteins, STRO-1, CD90 etc.), high replicative potential, high telomerase activity, and low expression of hypertrophy markers (Fellows et al., 2017; Matta et al., 2019; Nelson et al., 2014a; Williams et al., 2010). Since these cells are native to cartilage and possess progenitor like properties, they appear to be suitable for cartilage repair and inherently primed for chondrogenesis.

Although there are established protocols for isolation of pure populations of chondroprogenitors and chondrocytes, classical differentiating markers between the two cell populations have not been established. Our primary objective was to compare the cell types and evaluate differences in their biological characteristics based on flow cytometric analysis (FACS) to look for surface marker expression, RT-PCR for assessing markers of chondrogenesis and hypertrophy, tri-lineage differentiation to check for multipotency and comparison of growth kinetics. Since chondroprogenitors have been categorized as MSCs, the first category included markers of positive expression:CD105, CD73, CD90, CD106 and of negative expression:CD34, CD45 and CD14 (Dominici et al., 2006). The second category included markers considered to be expressed specifically by chondrocytes:CD54 (Kienzle and von Kempis, 1998) and CD44 (Knudson and Knudson, 2004). The final category included markers reported to be expressed by cells exhibiting enhanced chondrogenic potential:CD9 (Jayasuriya and Chen, 2015), CD29 (Koelling et al., 2009), CD151 (Fujita et al., 2006), CD49e (Williams et al., 2010), CD166 (Swart, 2002) and CD146 (Jiang et al., 2016; Su et al., 2015). To differentiate chondrocytes and chondroprogenitors on the basis of their chondrogenic potential and tendency for hypertrophy, mRNA expression for markers of chondrogenesis (collagen type II, aggrecan and SOX9), collagen type I and markers of hypertrophy (Collagen type X, RUNX2 and MMP-13) was analyzed.

Availability of OA cartilage is comparatively more than non-diseased cartilage and a plethora of knowledge exists regarding the effect of disease on cellular phenotype. Therefore, our second objective was to assess if OA differentially affects the cell populations, under consideration and cell samples isolated from non-diseased and OA human cartilage were compared. Our final objective was to study the effects of prolonged time in culture on the cell populations. This would afford additional information about chondrocyte behavior in culture and proposed potency of chondroprogenitors.

Section snippets

Study design

The study protocol was approved by the Institutional Review board and carried out in accordance to the guidelines laid down by the Institutional Ethics Committee. Human articular cartilage was harvested from three non-diseased (mean age:22 ± 4yrs, post-trauma above-knee amputations) and three OA (mean age: 63 ± 7yrs, undergoing knee replacement surgery) knee-joints. Written informed consent was obtained prior to sample collection. Our inclusion criteria for OA joints was primary degenerative OA

Growth kinetics

CFE ranged between 16%–25% for OA group and between 14%–17% for non-diseased group. When CPD was compared across cell groups at each passage from p0 to p10, there was no significant difference observed in the proliferative capacity between them, with all groups showing increase in their growth kinetics upto p6 (Fig. 4).

FACS

When surface marker expression was compared across each passage there was no significant difference observed between chondrocytes and chondroprogenitors derived from non-diseased

Discussion

Articular cartilage could be regenerated from its two native cell types (chondrocytes and chondroprogenitors), if their use in cell-based therapy is optimized. Extensive work on chondrocytes has afforded valuable information to their use in cartilage repair, although questions pertaining to their behavior in culture remain unanswered (Diaz-Romero et al., 2008; Hamada et al., 2013; Wang et al., 2004). On the other hand, chondroprogenitors, relatively recent in the field of cell-based repair have

Credit author statement

S.NoAuthor NamesCredit Roles
1Elizabeth Vinod
  • design of study

  • data curation and analysis

  • validation of data

  • writing

  • final approval of the manuscript

2Upasana Kachroo
  • design of study

  • data analysis

  • validation of data

  • writing

  • final approval of the manuscript

3Grace Rebekah
  • Formal analysis

  • Validation of data

  • Writing

  • Final approval of manuscript

4Bijesh Yadav
  • Formal analysis

  • Validation of data

  • Writing

  • Final approval of manuscript

5Boopalan Ramasamy
  • design of study

  • data analysis

  • validation of data

  • funding acquisition

  • writing

Funding information

This project was funded by AO Trauma Asia Pacific (AOTAP 16-19) of the AO foundation.

Declaration of Competing Interest

All author(s) declare(s) no conflict of interest

Acknowledgments

We thank the Centre for Stem Cell Research, (A unit of inStem, Bengaluru), Christian Medical College, Vellore, India for infrastructural support, Dr. Solomon Sathishkumar and Dr. George Thomas for critical revision of the manuscript.

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