Characterization of human articular chondrocytes and chondroprogenitors derived from non-diseased and osteoarthritic knee joints to assess superiority for cell-based therapy
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
design of study data curation and analysis validation of data writing final approval of the manuscript design of study data analysis validation of data writing final approval of the manuscript Formal analysis Validation of data Writing Final approval of manuscript Formal analysis Validation of data Writing Final approval of manuscript design of study data analysis validation of data funding acquisition writingS.No Author Names Credit Roles 1 Elizabeth Vinod 2 Upasana Kachroo 3 Grace Rebekah 4 Bijesh Yadav 5 Boopalan Ramasamy
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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