In vitro characterization of human articular chondrocytes and chondroprogenitors derived from normal and osteoarthritic knee joints

Objective Cell based therapy optimization is constantly underway since regeneration of genuine hyaline cartilage is under par. Although single source derivation of chondrocytes and chondroprogenitors is advantageous, lack of a characteristic differentiating marker obscures clear identification of either cell type which is essential to create a biological profile and is also required to assess cell type superiority for cartilage repair. This study was the first attempt where characterization was performed on the two cell populations derived from the same human articular cartilage samples. Design Cells obtained from normal/osteoarthritic knee joints were expanded in culture (up to passage 10). Characterization studies was performed using flow cytometry, gene expression was studied using RT-PCR, growth kinetics and tri-lineage differentiation was also studied to construct a better biological profile of chondroprogenitors as well as chondrocytes. Results and conclusions Our results suggest that sorting based on CD34(-), CD166(+) and CD146(+), instead of isolation using fibronectin adhesion assay (based on CD49e+/CD29+), would yield a population of cells primarily composed of chondroprogenitors which when derived from normal as opposed to osteoarthritic cartilage, could provide translatable results in terms of enhanced chondrogenesis and reduced hypertrophy; both indispensable for the field of cartilage regeneration.

(6,7) and fibrocartilage formation in vivo (8-10). Similarly, the limitations that affect chondrocyte 48 use (Autologous chondrocyte implantation) are graft hypertrophy and mixed fibro-hyaline 49 formation (11,12). Moreover, chondrocytes require expansion in-vitro, since cell yield post-50 harvest is too low to meet demands of direct implantation. This raises another conflict since there 51 does not appear to be a consensus on chondrocyte behavior in culture. There is evidence to show 52 that with increased time in culture, chondrocytes lose their phenotype and show higher expression 53 of markers for hypertrophy thereby reducing their efficiency for optimal cartilage repair(13,14). 54 However, there are also report which demonstrate that chondrocytes exhibit positive stem cell 55 markers in culture(15, 16). 56 Continued search for an optimal cell source led to a potential cell type residing within the  The second category for comparison included markers considered to be expressed specifically by  Since availability of OA cartilage is comparatively more than normal cartilage, and a dearth of   (Fig 1).

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Growth kinetics between the 4 groups was compared at each passage for estimation of cumulative 104 population doubling. IU/ml (Gibco) and amphotericin-B 2 μg/ml (Gibco). Medium was changed once every three days 118 (Fig 2). At a confluence of 85-90%, cell harvest was carried out using 0.25% Trypsin-EDTA 119 (Gibco). CPs were isolated from the superficial zone of cartilage (same source as used for chondrocytes).

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Alizarin red staining indicative of mineralization used to assess osteogenic potential showed 380 positive staining in both cell groups with higher uptake seen with chondrocytes (Fig 5C-D). Alcian expression albeit higher in chondrocytes though not to a significant level (Fig 6D). When 408 expression of negative MSC markers was assessed, we found that both cell population showed low 409 expression of CD45 and CD14 (Fig 7). However, there was a significant difference in the 410 expression of CD34 as levels in chondrocyte groups were much higher than CP groups (Fig 7A; 411 with prolonged expansion in culture even upto P10 (Fig 18). difference between the two (Fig 8) accord with an earlier study which suggests that CPs are a suitable contender for cell-based repair 454 due to their lower propensity for hypertrophy(44).

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In order to assess biological characteristics like replicative and differentiation potential which have 456 been reported to be superior in CPs (17)  in culture (up to p10) were used for acquiring information pertaining to parameters including 472 FACS, gene expression, replicative potential and tri-lineage differentiation. 473 We observe that both cell populations exhibit similar characteristics and since CD49e does not 474 seem to be a discrete marker of CP isolation, need for better suited bio-markers of differentiation 475 is warranted. Our findings suggest that cell sorting based on using CD34(-), CD166(+) and 23 476 CD146(+) will yield a population of cells primarily composed of CPs. This would be beneficial as 477 the results using RT-PCR and differentiation studies also indicate that CPs demonstrated a lesser 478 susceptibility for hypertrophy and a higher potential for chondrogenesis.

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In conclusion, the study implies that CPs derived preferably from normal as opposed to OA joints 480 and isolated using markers with higher specificity would yield translatable results in terms of 481 enhanced chondrogenesis and reduced hypertrophy, both indispensable for the field of cartilage 482 regeneration.