Migratory chondroprogenitors retain superior intrinsic chondrogenic potential for regenerative cartilage repair as compared to human fibronectin derived chondroprogenitors

Cell-based therapy for articular hyaline cartilage regeneration predominantly involves the use of mesenchymal stem cells and chondrocytes. However, the regenerated repair tissue is suboptimal due to the formation of mixed hyaline and fibrocartilage, resulting in inferior long-term functional outcomes. Current preclinical research points towards the potential use of cartilage-derived chondroprogenitors as a viable option for cartilage healing. Fibronectin adhesion assay-derived chondroprogenitors (FAA-CP) and migratory chondroprogenitors (MCP) exhibit features suitable for neocartilage formation but are isolated using distinct protocols. In order to assess superiority between the two cell groups, this study was the first attempt to compare human FAA-CPs with MCPs in normoxic and hypoxic culture conditions, investigating their growth characteristics, surface marker profile and trilineage potency. Their chondrogenic potential was assessed using mRNA expression for markers of chondrogenesis and hypertrophy, glycosaminoglycan content (GAG), and histological staining. MCPs displayed lower levels of hypertrophy markers (RUNX2 and COL1A1), with normoxia-MCP exhibiting significantly higher levels of chondrogenic markers (Aggrecan and COL2A1/COL1A1 ratio), thus showing superior potential towards cartilage repair. Upon chondrogenic induction, normoxia-MCPs also showed significantly higher levels of GAG/DNA with stronger staining. Focused research using MCPs is required as they can be suitable contenders for the generation of hyaline-like repair tissue.


Methods
Study design. All experimental protocols were approved by the Institutional Review Board (Research and Ethics Committee) of the Christian Medical College, Vellore. All methods were carried out in accordance with relevant guidelines and regulations.
After obtaining written informed consent from all subjects, human cartilage samples were harvested from three osteoarthritic (OA) knee joints of patients [age (mean ± SD): 55 ± 4 years], undergoing total knee replacement. These patients had high-grade osteoarthritis (Kellgren-Lawrence radiological score of 4). Patients whose knee joints had tumors, inflammation, or infection were excluded from the study. The cartilage slices were subjected to two different methods of isolation to obtain the individual chondroprogenitor subsets. Following enzymatic digestion, the released chondrocytes were subjected to selective adhesion assay using fibronectin to obtain progenitors. These subsets will hereon be referred to as fibronectin adhesion assay-derived chondroprogenitors (FAA-CP). Standard explant cultures were performed using cartilage slices taken from the surface of the knee joints. After 12 days of culture, outgrown cells were harvested and further cultured to obtain migratory chondroprogenitors (MCP). Both FAA-CP and MCP were expanded either under normoxia (21% O 2 and 5% CO 2 ) and hypoxia (1% O 2 and 5% CO 2 ). This study involved four groups for comparison, where FAA-CP and MCP were maintained in either normoxia or hypoxia culture conditions from their time of isolation to expansion and evaluation. The cells were expanded to passage 1 and subjected to characterization using fluorescence-activated cell sorting (FACS) for surface marker expression, studies using cell cycle analysis, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) for markers of chondrogenesis and hypertrophy, biochemical analysis of chondrogenic differentiated pellets for total GAG (glycosaminoglycan)/DNA content, and trilineage differentiation with confirmatory staining (Fig. 1). The experiments were conducted with three biological samples (n = 3).
Isolation and culture of FAA-CP. Cartilage slices from OA knee joints were washed and minced to a size of 1-2 mm. The FAA-CPs were isolated as per protocol previously described by Nelson et al. 15 In brief, the minced cartilage was subjected to sequential cellular digestion using pronase (12 IU, Roche) for 3 h followed by overnight incubation with collagenase type II (100 IU, Worthington) at 37 °C. The released cells were loaded on fibronectin-coated plates (10 µg/ml, Sigma) at a concentration of 4000 cells per well for 20 min. Following this, the non-adherent cells and medium were removed and replaced with stromal medium containing DMEM/ F12 (Sigma), 10% fetal bovine serum (FBS, GIBCO, Thermo Fischer Scientific), ascorbic acid (0.1 mM, Sigma), www.nature.com/scientificreports/ L-glutamine (2.4 mM/L, Sigma), antibiotics, and antimycotics. The adherent cells were maintained as previously described in either normoxic or hypoxic culture conditions for 12 days to obtain clones of > 32 cells (minimum population doubling of 5). The clones were isolated and replated at a ratio of 1 clone/cm 2 . Further expansion of enriched polyclonal chondroprogenitor cultures to passage 1 was done as per previously reported protocol 15 . The cells were expanded in a stromal medium containing human recombinant transforming growth factor beta2 (TGFβ2, Biovision) at 1 ng/ml, and human recombinant fibroblastic growth factor (FGF2, Biovision) at 5 ng/ml, as per standard established protocol.
Isolation and culture of migratory chondroprogenitors (MCP). Cartilage slices of 8-10 mm were harvested and placed in DMEM/F12 containing 10% FBS, 0.1 mM ascorbic acid, 10 mM Glutamine, antibiotics, and antimycotics (Fig. 2). Isolation was performed as per the protocol described by Koelling et al. and Wang et al 8,24 . In brief, following equilibration for a minimum period of 48 h the cartilage explants were placed in 0.1% collagenase type II (Worthington) for a period of 2 h at 37 °C. Further, the cartilage slices, devoid of any released cells were washed, and placed in a culture plate containing to stromal medium in a culture plate and observed for the outgrowth of cells. After 10 days, the migrated cells were harvested and further expanded to passage 1 for further characterization. For all cultures, the medium was changed once every three days, and at sub-confluence (85-90%), cell harvest was carried out using 0.125% Trypsin-EDTA (GIBCO, Thermo Fischer Scientific). It was ensured that the culture, expansion, and differentiation of the four groups (FAA-CP vs MCP, normoxia vs hypoxia) were performed under their specified culture conditions. Since the standardized culture conditions recommend the requirement of additional growth factors for FAA-CPs 14,15,32 but not for MCPs 8,34,35 , the two types of chondroprogenitors were cultured accordingly.
Cell diameter and cell cycle analysis. The percentage of cells in different phases of the cell cycle was analyzed using DAPI (4' ,6-diamidino-2-phenylindole). At a confluence of 60-70% and a day after medium change, the cells were trypsinized. Cell size estimation was done using an automated counter (Countess, Invitrogen). Passage 1 chondroprogenitors at a concentration of 1 × 10 6 cells were washed with phosphate buffer solution (PBS) and fixed with cold 70% ethanol for 1 h. Following a wash, DAPI at a concentration of 1 µg/ml (in PBS containing 0.1% TritonX100) was used for incubation for a duration of 30 min. The washed cells were resus-  Table S1). Data acquisition was done using BD FACS Cytoflex cytexpert. Unstained controls were run in parallel, and data were analyzed using BD FACS Diva v 8.0.1.1 and Flo Jo software.

Quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The harvested cells
were washed in PBS and subjected for total RNA extraction using Qiagen RNeasy Mini Kit as per the manufacturer's instruction. The absorbance at 260 nm and 280 nm (A260/A280) was used to determine the RNA concentration using a nanodrop spectrophotometer. Complementary DNA was synthesized with 280 ng of RNA, using the Takara Bio First-Strand synthesis system. PCR assays were carried out using Takyon Low Rox SYBR Master Mix Dttp Blue (Eurogentec, Belgium) on a QuantStudio 6 K Flex thermocycler (Applied Biosystem). The expression of chondrogenic marker genes (SOX-9, ACAN, and COL2A1), fibrocartilage marker gene COL1A1, and hypertrophic chondrocyte marker genes (COL10A1, RUNX2, and MMP-13) was measured. The relative mRNA expression for all target genes was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to obtain the ΔCt. GAPDH was utilized as the housekeeping gene as it demonstrated high and stable expression under varying conditions (data not shown). The obtained ΔCt values were compared to the normoxia FAA-CP group (ΔΔCt), from which 2^− ΔΔCt was calculated. In addition, the ratio of COL2A1 to COL1A1 was calculated to evaluate their hyaline cartilage forming potential. The experiments were conducted with three biological samples (n = 3) in two technical replicates. Specific primer sequence (5'-3' and 3'-5'), accession code, gene identifier, and base-pair length for the eight genes are listed in Supplementary Table S2.
Multilineage differentiation and confirmatory staining. Induction towards adipogenic, osteogenic, and chondrogenic lineage was performed using StemPro differentiation kits (Thermo Fischer, Cat no: A1007201, www.nature.com/scientificreports/ A1007001, and A1007101). Before adipogenic and osteogenic differentiation, cells were seeded at a concentration of 5000 cells/cm 2 in 24 well culture dishes and expanded to sub confluence. Then, the stromal media was replaced with the differentiation media. The control arm included cells grown in a stromal medium for the same period. For three-dimensional pellet cultures, 0.5 × 10 6 cells were loaded into Eppendorfs, centrifuged at 400 g for 12 min, and left uninterrupted for 24 h. Following this, StemPro chondrogenic medium was added to the pellets. The medium was changed every three days for a period of three weeks.
Adipogenic and osteogenic staining. Differentiated adipocytes were fixed for 1 h with 10% formaldehyde, washed, and stained with Oil Red O (Sigma), while differentiated osteocytes were fixed for 1 h with 70% ethanol, washed, and stained with Alizarin Red (Sigma). Parallel controls were also subjected to staining and imaging using the Olympus virtual slide system.
Chondrogenic staining. Following differentiation, the pellets were washed and fixed with 4% paraformaldehyde for a period of 15 min. The cassettes containing the pellets were dehydrated for 10 min in a series of ethanol solutions of increasing concentration (70 to 100%). This was followed by ethanol clearance using xylene, wax infiltration, and paraffin embedding into mold cassettes. Paraffin sections (4 µm) were obtained using a microtome on poly-L lysine (PLL) coated slides and kept at 65 °C for 2 h. The sections were hydrated with descending grades of alcohol (100% to 70%) and washed prior to processing. Biochemical analysis: total GAG/DNA content of chondrogenic pellets. For quantification of GAGs and DNA, pellets were collected 3 weeks after chondrogenic induction and digested with 120 µg/ml papain at 65 °C for 16 h. DNA content was measured by Quant-iT Picogreen dsDNA reagent, and Lambda DNA was used for the generation of the standard curve. Fluorescence intensity was measured using the SpectraMax i3x Reader (Norwalk, CT, USA) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. GAG content was then quantified using the dimethyl methylene blue dye method according to the manufacturer's instructions (Chondrex, Cat No: 6022). Chondroitin 6-sulfate was used as a standard. The optical density was measured at 525 nm and total GAG content was calculated. For comparison of GAG production, GAG content was normalized to DNA content (GAG/DNA). Statistical analysis. Data analysis was performed using SPSS version 21.0 (SPSS Inc, Chicago, USA) and graphical presentation using Prism v6 (GraphPad). The One-way ANOVA with post hoc LSD correction was used to assess the significance of differences between the four groups. Numerical values were expressed as mean ± standard error mean. A P value of < 0.05 was considered to be significant.

Results
Cell diameter and cell cycle analysis. Both normoxia and hypoxia FAA-CPs demonstrated clonal growth, achieving a population doubling of 5 by day 12 (Fig. 2a,b). Concerning MCPs, migration of chondroprogenitors from cartilage explants was observed as early as day 7 (Fig. 2d). On further expansion, both the cell groups displayed spindle-shaped morphology with a honeycomb pattern of growth, a hallmark of the MSC population ( Fig. 2e-h). When the average cell diameter (µm) of passage 1 cells (n = 3) were compared, there was no significant difference observed between the groups (Fig. 3a). Analysis of the different phases in cell cycle distribution between the groups showed that both FAA-CPs and MCPs expanded under hypoxia displayed a greater percentage at G1 interphase and lower percentage at G2M interphase as compared to their normoxic counterparts. However, when the DNA synthetic S phase was compared the highest values were observed with normoxia FAA-CPs. The observed differences were not significant, though the MCPs were expanded in a stromal medium devoid of additional growth factors (Fig. 3b). www.nature.com/scientificreports/ Surface marker expression using FACS. Flow cytometric analysis of the two populations was performed to study surface marker expression based on the mentioned categories. When MSC markers were compared, all the groups (n = 3) displayed a high expression of positive markers CD105, CD73, and CD90, and a low expression of negative markers CD34, CD45, and CD14 (Table 1). There was no significant difference between the groups except for CD105, which was higher in normoxia MCP when compared to both normoxia and hypoxia FAA-CP (P < 0.01, Fig. 3c). Regarding the integrin markers, all the groups showed a high expression of CD49e and CD29, with no significant difference, except for CD49b, which was higher in the normoxia MCP group as compared to FAA-CPs (P < 0.05, Fig. 3d). The hypoxia MCP showed higher levels of CD49b when compared to the hypoxia FAA-CP group alone (P = 0.036). When potential markers of chondrogenesis were assessed, all the groups demonstrated high and comparable levels of expression (Table 1). Concerning the immunogenic markers, all the groups displayed a comparable and positive expression of HLA-ABC, with a low expression of MHC II and its co-stimulatory molecules. www.nature.com/scientificreports/ qRT-PCR. With reference to markers of chondrogenesis, a high mRNA expression of ACAN and a moderate expression of SOX-9 and COL2A1 was seen in all the groups with no significant difference between them, except for ACAN (Fig. 4). Normoxia MCP displayed a stronger expression of the gene ACAN when compared to normoxia FAA-CP (P = 0.027). An analysis of the markers of hypertrophy showed differences between the two chondroprogenitor groups. A moderate to low expression of COL10A1, RUNX2, and MMP13 was observed in all the groups (Fig. 4). An intergroup comparison revealed that the MCPs showed remarkably lower levels of COL1A1 (fibrocartilage marker) and RUNX2 (key transcription factor for the genesis of osteoblast) when compared with FAA-CPs [(a) COL1A1: normoxia MCP Vs FAA-CPs, P = 0.000, hypoxia MCP vs FAA-CPs, P < 0.005, (b) RUNX2: normoxia MCP Vs FAA-CPs, P = 0.024, hypoxia MCP vs FAA-CPs, P = 0.009]. Only hypoxia MCPs exhibited significantly lower levels of COL10A1, another marker of osteogenesis and hypertrophy, as compared to normoxia FAA-CPs (P = 0.002). A comparison of normoxia FAA-CPs and MCPs to their hypoxia counterparts showed that hypoxic conditions reduced the expression of COL10A1 in both groups (P = 0.030 and P = 0.014 for FAA-CP and MCP respectively). Additionally, normoxia MCPs showed decreased levels of COL1A1 than hypoxia MCPs (P = 0.013). Although there was no difference seen with the expression of the mature type II collagen between the groups, the functional COL2A1/COL1A1 ratio was significantly increased in normoxia MCPs as compared to the other three groups (P < 0.05). Overall, we observed that normoxia MCPs showed higher levels of chondrogenic genes and lower levels of hypertrophic markers. As described in the methods section, the FAA-CPs and MCPs were cultured using standard culture media recommended and reported for these cells in previous studies.
Multilineage differentiation, confirmatory staining, and total GAG/DNA content. All the study groups demonstrated trilineage differentiation potential. When adipogenic and osteogenic potentials were qualitatively assessed using Oil Red O (lipid droplet accumulation) and Alizarin red S (calcified matrix deposition) respectively, all groups displayed positive staining (Fig. 5). Routine histological analyses of the pellets following chondrogenic differentiation showed that all the groups displayed comparable staining for Picrosirius red (Fig. 6e-h), except for normoxia MCPs, which displayed a stronger uptake for Alcian blue and Toluidine blue ( Fig. 6a-d, i-l). Immunohistochemical staining for Collagen type II protein showed positive expression intensity in all the groups, with no apparent difference between them (Fig. 6m-p). When the chondrogenic induced pellets were digested and the DNA and GAG contents were measured, the normoxia MCPs demonstrated significantly higher levels of GAG/DNA as compared to the normoxia FAA-CP (P = 0.001), the hypoxia FAA-CP (P = 0.002), and the hypoxia MCP (P = 0.009) (Fig. 7, Supplementary Table S3). The higher levels of extracellular matrix (ECM) synthesis, observed with normoxia MCP, corroborated with the gene expression analysis for chondrogenesis.

Discussion
The clinical goal of cartilage repair strategies is to produce a repair tissue that has identical functional and mechanical qualities as the hyaline articular cartilage. Though a multitude of reports using cell-based therapy has demonstrated therapeutic efficiency, the ensuing fibrocartilage tissue tends to be a poor substitute for longterm subsistence 39 . The discovery of cartilage resident progenitors, displaying superior chondrogenic potential and lower hypertrophic tendencies, has led to the evaluation of their use for tissue engineering applications 23 . However, clarification of their biological properties is warranted before their therapeutic use can be considered.
The main goal of this study was to compare the biological properties of resident cartilage progenitors which were derived using two distinct, but standard methods of isolation and expansion, focusing on their prospects for cartilage repair. We compared FAA-CPs and MCPs and evaluated their properties based on growth kinetics, immunophenotyping, and multilineage differentiation capacity under normoxic and hypoxic culture conditions. Further, the chondrogenic commitment was also compared, by analyzing the matrix deposition and gene expression of common chondrogenic and hypertrophic markers.
There exists a positive correlation between morphological cellular changes and chondrogenesis 40 . Spindleshaped and fibroblast-like chondroprogenitors transform into spherical chondrocyte-like cells 41 . The changes that occur at the cellular level are the result of events that occur at the molecular level. Our results show that MCPs displayed similar morphological features and cell cycle patterns as FAA-CPs, even in the absence of additional exogenous growth factors. The isolation of chondroprogenitors in both groups required only a minimal quantity of starting cartilage material. The antigen expression pattern showed that both chondroprogenitor groups complied with the minimal ISCT criteria for MSCs with normoxia MCPs displaying significantly higher www.nature.com/scientificreports/ levels of CD105. CD105, in addition to being a marker for MSC, has also been reported to be a predictive marker for chondrogenesis [42][43][44][45] . With regard to the integrin markers, the MCPs displayed comparable levels of the heterodimeric fibronectin receptor (CD49e/CD29), even without treatment to the fibronectin surface. This result corroborated with a recent report that questioned the specificity of the integrin as a distinctive marker for migratory chondroprogenitors 46 . However, the expression of CD49b was higher in MCPs, similar to a recent   www.nature.com/scientificreports/ report categorizing them to be distinctive for MCPs 47 . CD146, a known marker of enhanced chondrogenesis, was notably higher with the MCP groups, though not significant due to a single donor variance. Cell proliferation in eukaryotic cells is controlled by the precise transition from G1 to S phases of the cell cycle 48 . We observed that hypoxia groups displayed higher G1 percentages than their normoxia counterparts, however, only normoxia FAA-CPs presented the highest occurrence at the S phase. Concerning G2M phase occurrence 49 , hypoxia reduced the arrest in this phase indicating their preponderance for increased cell proliferation. These finding though not statistically significant necessitates further in-depth evaluation to understand the complex mechanisms that regulate the temporal order of transcription activation and inactivation for the distinct groups. SOX-9 and RUNX2 are the principal regulators of chondrogenesis with different functional roles. SOX-9 plays a vital role in articular cartilage formation, while RUNX2 is primarily involved in the hypertrophic maturation of cells 50 . Both transcription factors are expressed through the entire process of chondrogenesis, starting from mesenchymal condensation and ending with terminal chondrocyte hypertrophy. Aggrecan and type II collagen protein, being the major components of the articular cartilage ECM, have also been categorized as markers of chondrocyte phenotype for in-vitro studies 51 . Thus, when considering FAA-CPs and MCPs for their distinct use in cartilage regeneration, a number of variations were observed between the two cell populations. The molecular analysis of normoxia MCPs showed greater expression of the chondrogenic markers SOX-9 and COL2A1, with a significantly higher expression with ACAN and COL2A1/COL1A1 ratio. These findings corroborate with other studies reporting higher expression of primary chondrogenic transcription factor SOX9 and stemness marker namely KLF4 in adipose-derived MSCs 52 and increased proliferation of MSCs 53 grown under normoxia as compared to hypoxic cultures. The RT-PCR comparison should be viewed in light of the fact that the undifferentiated cells were grown with their recommended standard media with growth factors as per their requirement, and not under a single formulation. Furthermore, an analysis of the GAG/DNA ratio obtained from chondrogenically differentiated pellets showed that normoxia MCPs outperformed the other cell groups, additionally displaying stronger uptake for glycosaminoglycans with Alcian blue and Toluidine blue.
Among the several molecular transcription factors reported to induce hypertrophy of chondrocytes leading to cell apoptosis and endo-vascularization, RUNX2 plays a major role 54,55 . Additionally, hypoxia has been reported to suppress terminal differentiation of chondrocytes with evidence of hypoxia-inducible factor-α repressing the expression of RUNX2. An evaluation of the limitation to hypertrophic commitment showed that, regardless of the microenvironment, both normoxia and hypoxia MCPs showed significantly lower levels of RUNX2 and COL1A1. Although hypoxic conditions did not induce a significant difference in the expression of chondrogenic genes, it favoured a significant decrease of COL10A1 56 , a known biomarker of terminally differentiated and hypertrophic chondrocytes, within the FAA-CP and MCP groups respectively. In accordance with our findings, a recent study demonstrated that equine MSCs isolated from synovial membrane and bone marrow showed reduced levels of COL10A1 when cultured under hypoxic conditions without any effect on chondrogenesis markers 57 . When results for trilineage differentiation were compared, a positive uptake with a similar pattern of staining was observed among all the groups, showing comparable differentiation potential.
The work reported in this study provides the first comprehensive comparison of the chondrogenic potential of human FAA-CPs and MCPs, in addition to investigating the effect of hypoxia on chondrogenesis. This study is also the first attempt to evaluate the properties of two types of progenitors derived from the same source of cartilage, and also utilizing human biological tissue. Although our results suggest that migratory chondroprogenitors cultured under normoxic conditions demonstrate higher chondrogenic potential and limited commitment to hypertrophy, hypoxia cultures display lower levels of collagen type 10, a finding that merits further evaluation to www.nature.com/scientificreports/ understand their additional implications for chondrogenesis. Thus, further in-depth surfaceome and proteomic analysis would help understand the beneficial effect of varying culture conditions on chondrogenesis. Since the chondroprogenitors were isolated from osteoarthritic samples, the inflammatory microenvironment could have influenced their biological properties. Further evaluation using cells obtained from non-diseased joints, using similar media formulations, and revalidation using in-vivo study models would merit further understanding of their potential therapeutic application in the field of cartilage repair.