Pondering the Potential of Hyaline Cartilage–Derived Chondroprogenitors for Tissue Regeneration: A Systematic Review

Abstract

Objective

Chondroprogenitors have recently gained prominence due to promising results seen in in vitro and animal studies as a potential contender in cell-based therapy for cartilage repair. Lack of consensus regarding nomenclature, isolation techniques, and expansion protocols create substantial limitations for translational research, especially given the absence of distinct markers of identification. The objective of this systematic review was to identify and collate information pertaining to hyaline cartilage–derived chondroprogenitors, with regard to their isolation, culture, and outcome measures.

Design

As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a web-based search of Scopus and PubMed databases was performed from January 2000 to May 2020, which yielded 509 studies. A total of 65 studies were identified that met the standardized inclusion criteria which comprised of, but was not limited to, progenitors derived from fibronectin adhesion, migrated subpopulation from explant cultures, and single-cell sorting.

Result

Literature search revealed that progenitors demonstrated inherent chondrogenesis and minimal tendency for hypertrophy. Multiple sources also demonstrated significantly better outcomes that bone marrow–derived mesenchymal stem cells and comparable results to chondrocytes. With regard to progenitor subgroups, collated evidence points to better and consistent outcomes with the use of migratory progenitors when compared to fibronectin adhesion assay–derived progenitors, although a direct comparison between the two cell populations is warranted.

Conclusion

Since chondroprogenitors exhibit favorable properties for cartilage repair, efficient characterization of progenitors is imperative, to complete their phenotypic profile, so as to optimize their use in translational research for neocartilage formation.

Introduction

Optimization of cell-based therapy is essential to alleviate the immense global burden of cartilage afflictions. Chondroprogenitors have recently garnered interest as a potential contender for cartilage repair due to their phenotypic tendency toward chondrogenesis and minimal hypertrophy. ¹ Additionally, they have been likened to multipotent mesenchymal stromal cells/mesenchymal stem cells (MSCs), demonstrating features as described by the International Society for Cellular Therapy (ISCT) criteria 2006, such as adherence to plastic, specific surface marker expression and multilineage potential. ² In the sequence of chondrogenesis, MSCs condense and aggregate to form progenitors, which differentiate further to form chondrocytes. ³ This lineage bias exhibited by chondroprogenitors can prove to be useful for both cell-based and tissue engineering strategies for neocartilage formation. However, lack of clarity regarding the definition of said progenitors, coupled with multiple methods of isolation, render categorization imprecise. Previous literature reports chondroprogenitor isolation from various tissue sources such as articular cartilage, synovium, meniscus, and infrapatellar fat pad, among others.^4,5 As the end goal remains regeneration of hyaline-like cartilage, accurate characterization, and optimization of chondroprogenitors isolated from hyaline cartilage, is imperative and may prove to be beneficial in the long run since they are naturally primed for chondrogenesis.

Historically, chondroprogenitors were first detected by Hayes et al. as label-retaining cells in the superficial zone of fetal cartilage. ⁶ Circumstantial evidence demonstrating presence of these cells driving appositional growth led Dowthwaite et al. to isolate and characterize chondroprogenitors, derived from superficial layer of articular cartilage using fibronectin adhesion assay. ⁷ In addition to these early studies, characterizing of progenitors demonstrated high SRY box transcription factor 9 (SOX9) and Notch homolog 1 (NOTCH1) expression, affinity for CD49e, and higher telomerase activity when compared to mature chondrocytes.^8,9 Alternatively, Koelling et al. studied the potential role of chondroprogenitors with respect to homing/migration in response to cartilage injury, thus contributing to tissue repair. ¹⁰ This method of isolation of progenitors based on their migratory potential yielded cells that also displayed high chondrogenic potential and features similar to MSCs. Another variation utilized for selection of chondroprogenitors from the general pool of chondrocytes, is based on segregation using potential chondrogenic surface markers. Studies detailing cellular sorting used to obtain progenitors have also reported higher migratory potential and chondrogenic potential in the final isolate.^11,12

This systematic review focused on information pertaining to chondroprogenitors isolated from hyaline cartilage, namely nasal septum, and articular surface. All published literature spanning the previous 20 years concerning hyaline cartilage derived chondroprogenitors, isolated via fibronectin adhesion assay, cartilage explants (migratory progenitors), and single cell sorting was assessed and compared. In summarizing available reports, evaluation of cell type most suitable for cartilage repair was performed. This was based on comparison with routinely used cells (MSCs and chondrocytes) and consistency in measured outcomes especially in terms of chondrogenic potential.

Methods

Search Strategy

The literature review was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) statement. An electronic search was performed on “Scopus” and “PubMed” database for the duration spanning January 2000 to May 2020. Due to the diverse nomenclature of the term chondroprogenitors, the search was carried out using the following keywords separately: “chondroprogenitor,” “cartilage-progenitor,” and “chondrogenic-progenitor.” Inclusion criteria comprised all studies published in English language which involved isolation of chondroprogenitors from hyaline cartilaginous tissue using (a) fibronectin adhesion assay, (b) migratory subpopulations derived from cartilage explants, and (c) cell sorting techniques. Reviews, studies involving chondroprogenitors derived from non-cartilaginous tissue (such as differentiated bone marrow mesenchymal stem cells [BM-MSCs], induced pluripotent stem cells [iPSCs], etc.) or using chondroprogenitor cell lines (e.g., ATDC5) were excluded. Two independent observers (EV and UK) conducted the screening process and analysis, following which 2 authors reanalyzed the collated data (RP and BR). First the titles and abstracts were screened using the selection criteria, as mentioned above and further eligibility was confirmed after reviewing the full text. A final screening of the listed articles was also performed to eliminate duplicated publications.

Results

The search database identified a total of 358 articles on Scopus and 151 articles using PubMed. After eliminating 115 paper duplicates, 394 articles were analyzed against the inclusion criteria. After exclusion of 329 articles, the remaining 65 were included for qualitative analysis ( Figure 1 ). A standardized format was used for chronological tabulation of data obtained from the reports analyzed. Categories included source of cartilage sample (species), method of isolation and culture (culture medium used, seeding density, and study timeline), assessment parameters, and study outcomes.

Figure 1.

Summary using Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) flow literature search diagram.

Fibronectin Adhesion Assay–Derived Chondroprogenitors

Source of Tissue

A total of 43 reports described chondroprogenitors isolated via fibronectin adhesion assay ( Table 1 ). Cartilage specimens were sourced from both human donors (n = 21) and animals (n = 23). Animal sources included bovine (n = 8), equine (n = 6), swine (n = 2), leporine (n = 2), and murine (n = 5). Literature search revealed that the cartilage samples were collected from the following anatomical sites: knee joint (n = 21), metacarpophalangeal (n = 9), metatarsophalangeal (n = 3), and nasal septum (n = 2), whereas in 8 reports the site of origin was not mentioned. Human cells were derived from either osteoarthritic (n = 10) or nondiseased (n = 7) articular cartilage. Eight reports went on to perform in vivo experiments with chondroprogenitors, while the rest were limited to in vitro investigations.

Table 1.

Summary of Cartilage–Derived Chondroprogenitors Using Fibronectin Adhesion Assay from Articular Hyaline Cartilage.

S. No.	Method of Isolation	Cartilage: Species	Isolation Culture condition				Objectives	Outcome Measures	Results
	Fibronectin Adhesion Assay (FAA)		Cartilage Slices: Sequential Digestion Isolation of CPs		Culture Medium and Additives	Seeding Density and Experimental Time			ISCT Criteria	Reported CD Markers	Differentiation Potential	Overall
	Study: Reference		Pronase	Collagenase
1	Dowthwaite et al., 2004 7	7-day old calves	700 IU/mL + 10% FCS: 15 min to 1 h	Type I 300 IU-900 IU/mL: 3 h	DMEM/F12 + 10% FCS • 0.5 µg/mL ascorbate • 1 µg/mL/glucose	FAA (10 µg/mL) 4000 cells/mL: 20 min to 40 min 10 days	Isolate and characterize CPs from surface zone of cartilage	FACS: α5, β1, NOTCH1 Immuno-cytochemistry: Fibronectin, NOTCH1 Notch transfection	N	CD29 CD49	Adipogenic Osteogenic Chondrogenic	Express NOTCH1 High fibronectin (α5β1) affinity and CFE
2	Martin et al., 2005 20	Bovine metatarsal phalangeal joints	0.1%: 3 h	Type I 0.04%: overnight	DMEM + 10% FCS + 2 mm L-glutamine + 1% nonessential AA Variables: • FCS (10% to 40%) • TGβ1 (1-10 ng/mL) • FGF2 (1-10 ng/mL) Cryopreservant: FCS + 10% DMSO	FAA (10 µg/mL): 4,000,cells/mL 0 min; 12 days Expansion: 104 cells/cm2; sub-confluence Cryovial: 2 × 106 CP/vial	Cryopreservation and in vitro expansion on CP survival, proliferation, differentiation ability, substrate limitation, contact inhibition	Cell density, viability, COL II, Saf-O	N	—	—	Post cryo-viability: 14 days: 92.99% Optimal medium: 40% FCS +1 ng/mL TGFβ1
3	Melero-martin et al., 2006 18	Bovine metatarsal phalangeal joints	0.1%: 3 h	Type I 0.04%: overnight	DMEM + 10% FCS + 2 mm L-glutamine + 1% nonessential AA Variables: • Standard (10% FCS, 48 h) • High FCS feeding (40% FCS, 12 h) • Optimized batch (40% FCS, TGF-β1: 1 ng/mL)	FAA (10 µg/mL): 4,000 cells/mL 20 min; 12 days Expansion: 104 cells/cm2	Mathematical approach: optimize seeding density and passage length: semi-algorithmic data plots	Initial seeding density, passage length	N	—	—	Optimal seeding density: 104 cells/cm2 Passage length: 73 h with optimized batch at 0.3 mL/cm2 of medium
4	Melero-Martin et al., 2006 19	Bovine metatarsal phalangeal joints	0.1%: 3 h	Type I 0.04%: overnight	DMEM + 10% FCS + 2 mm L-glutamine + 1% nonessential AA Variables: • 10% FCS • 40% FCS • 10% FCS + TGF-β1: 1 ng/mL • 40% FCS + TGF-β1: 1 ng/mL	FAA (10 µg/mL): 4,000 cells/mL 20 min; 12 days Expansion: 104 cells/cm2 CultiSpher-G: 16.7 mL medium + 1.8 g/L microcarriers	Macroporous Microcarrier culture system (CultiSpher-G)	Cell density, viability, COL II, MTT, SEM	N	—	—	Achieved cell density: 5.5 × 105 cell/mL 17-fold expansion with 40% FCS + TGF-β1 than monolayers
5	Ustunel et al., 2006 28	Human	3.17 U/m: 3 h	Type II 0.12 U/mL	DMEM + 10% FCS	FAA (10 µg/mL): 4,000 cells/mL 20 min; 12 days	IHC localization of Notch receptors and its ligands in comparison to FAA derived clones in vitro	IHC: Notch1-4 Delta Jagged1-2 SEM TEM	N	—	Chondrogenic	Co-relational presence of Notch1,2 and Jagged-1 CPs form larger colonies than chondrocytes
6	Khan et al., 2009 8	Juvenile bovine metacarpal phalangeal	Not reported	Not reported	DMEM + 10% FCS	FAA (10 µg/mL): 700 cells/mL-20 min Expansion: Enriched polyclonal cultures up to 2 weeks	Characterize and understand CPs growth kinetics and phenotype	Growth kinetics: cell count, telomere length, telomerase activity, β-galactosidase, RT-PCR: SOX9, ACAN	N	—	Chondrogenic	Exponential growth for 20PD Greater telomerase activity (2.6-fold), SOX9 than chondrocytes
7	Williams et al., 2010 9	Nondiseased human femoral condyle	70 IU/mL: 1 h	Type I 300 IU/mL: 3 h	DMEM/F12 + 10% FCS • 0.1 mM ascorbate-2-phosphate • 0.5 mg/mL L-glucose • 2 mM L-glutamine • 10 mM HEPES • TGβ1 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 4,000 cells/mL-20 min; 12 days Expansion: >3 clones/specimen and enriched polyclonal cultures up to p5 Cell label: PKH26: 2 × 107 cells/mL	In vitro isolation and characterization of articular CPs In vivo caprine chondral defect	FACS, IHC, differentiation assay, cytogenetic analysis, telomere length Caprine in vivo study	N	CD49e CD166 CD105 CD29	Adipogenic-RT-PCR: lipoprotein lipase Osteogenic: limited Chondrogenic	Classified MSC with CD49e affinity High telomerase and maintain telomere length Abnormal karyotype Excellent integration of chondral defect
8	McCarthy et al., 2012 1	Equine metacarpal phalangeal joints	700 IU/mL	300 IU/mL	DMEM/F12 with GlutaMAX-1 + 10% FCS • Ascorbate-2-phosphate • 10 mM HEPES • FGF2 (10 ng/mL)	FAA (10 µg/mL): 4,000 cells/mL-20 min; 6 days Expansion: Enriched polyclonal cultures up to confluence	Compare equine CPs and BM-MSCs for cartilage repair	IHC: CD166, STRO-1, Notch-1, CD90 Differentiation studies Hypertrophy markers: COL X, matrilin-1, RUNX2	N	CD90 CD166	Adipogenic Osteogenic Chondrogenic	Both populations demonstrated putative stem cell markers CPs lower levels of hypertrophic markers
9	Ozbey et al., 2014 29	Human femoral condyle	70 IU/mL: 1 h	300 IU/mL: overnight	DMEM/F12 + 10% FCS • 0.1 mM sodium ascorbate • 0.5 mg/mL L-glucose • 2 mM L-glutamine • 1 mM sodium pyruvate • 100 mM HEPES	FAA (10 µg/mL): 4,000 cells/mL-20 min; 10-15 days Expansion: Enriched polyclonal cultures up to p3	Determine localization of putative stem cell markers in cartilage tissue and expanded CPs	IHC: STRO-1, Oct3/4, antI-CD90, anti-CD105, anti-CD166 Differentiation studies	N	CD105 CD166 CD90	Adipogenic Osteogenic Chondrogenic	Putative stem cell markers present even at deeper zones with low Oct3/4 And capable of differentiation even at p3
10	Nelson et al., 2014 69	Human OA tibial plateau	70 IU/mL	300 IU/mL	DMEM/F12-Glutamax + 10% FCS • 50 µg/mL l-ascorbic acid; 2-phosphate • 1 mg/mL: 1 glucose • 2 mM l-glutamine	FAA (10 µg/mL): 4,000 cells/mL-20 min; 8-14 days	Isolate CPs from OA cartilage and evaluate stem cell characteristics	CFE Growth kinetics Differentiation potential: staining, RT-PCR	N	—	Adipogenic Osteogenic Chondrogenic	OA CPs display trilineage potential CFE <0.1% Clonal variations suggestive of CP subpopulations
11	Marcus et al., 2014 48	Juvenile bovine metacarpal phalangeal	3.17 U/mL: 3 h	Type I 0.12 U/mL: overnight	DMEM + 10% FCS	FAA (10 µg/mL): 4,000 cells/mL-20 min; 5 PD Expansion: Single clone and enriched polyclonal cultures up to sub confluence Cell label: PKH26: 2 × 107 cells/mL	Chondrogenic potential and functional matrix production of CPs injected in SCID	Histology/RT-PCR: Saf-O COL II SOX9	N	—	Chondrogenic	Detectable post-implantation Failed: robust cartilage pellet despite SOX9, COL II expression
12	Xue et al., 2015 30	Hybrid piglets	—	0.2%: 8 h	Low-glucose DMEM/F12 + 10% FCS	FAA (10 µg/mL): 4,000 cells/mL-20 min; 14 days Expansion: 2,000 cells/cm2	Comparison of chondrogenic potential of BM-MSC, auricular, intervertebral and articular cartilage derived CPs	CFE Cell proliferation and differentiation RT-PCR: ACAN, SOX9, COL II	N	CD29 CD90 CD35 CD34 CD45	Adipogenic Osteogenic Chondrogenic	Cell lines were CD29 and CD90 positive Articular cartilage derived CPs showed higher expression of chondrogenic genes than BM-MSC
13	Frisbie et al., 2015 45	Equine knee joints: lateral trochlear ridge	70 IU/mL + 10% FCS: 15 min	Type Ia 300 IU/mL: 3 h	DMEM/F12 + 10% FCS • 0.1 mM ascorbate-2-phosphate • 0.5 mg/ml L-glucose • 10 mM HEPES • TGβ1 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 4,000 cells/mL-20 min; 6 days Expansion: Monoclonal CPs up to 14 days Articular defect: 36 × 106 CPs (600 mL fibrinogen + 600 mL thrombin)	Autologous and allogenic CPs in fibrin for articular defect(15 mm) healing	Musculoskeletal examinations Femoropatellar radiograph IHC: COL II, COL X	N	—	Chondrogenic	Autologous CPs better repair than allogeneic and controls
14	Neumann et al., 2015 70	Nondiseased human (tibial plateau)	70 U/mL: 1 h	Type I 300 U/mL: 3 h	DMEM-Glutamax + 10% FCS • 0.1 mM ascorbic acid • 0.5 mg/mL L-glucose • 10 mM HEPES • 1 mM sodium pyruvate • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 4,000 cells/mL-20 min; 12 days Expansion: Polyclonal: 13,333 cells/cm2 1st up to 26 PD 2nd + 7.6 PD	CPs response: mechanical stimulation/adenoviral-mediated overexpression of BMP-2	3D Fibrin/polyurethane Hoechst 33258 dye assay IHC: COL II, COL X Transduction BMP2	N	—	Chondrogenic	Mechanical stimulation increased chondrogenic genes BMP2 increased hypertrophy markers
15	Shafiee et al., 2016 31	Human nasal septum	0.2%: 1 h	Type I, II and dispase: 16 h	Low-glucose DMEM + 10%FCS	FAA (10 µg/mL) 4,000 cells/mL: 20 min	Comparison of chondrogenic potential between BM-MSC, AD-MSC, CPs and nasal septum CPs	Cell cycle and karyotype analyses FACS Nano-scaffold chondrogenic differentiation potential	N	CD73 CD90 CD105 CD106 CD166 CD34 CD45 HLA-DR HLA-ABC	Chondrogenic	Similar phenotypic expression Nano scaffold supported differentiation of all cell types Nasal cartilage progenitors showed highest proliferative and chondrogenic potential
16	Li et al., 2016 21	Rabbit knee joints	700 IU/mL + 10% FCS	Type I Not specified	DMEM + 10% FCS	FAA (10 µg/mL) 4,000 cells/mL; 10 days	Intermittent hydrostatic pressure on chondrogenic potential of CP vs. IPFSC in alginate beads	GAG, Saf-O, DMMB IHC: COL II	N	CD90 CD105 CD166	Adipogenic Osteogenic Chondrogenic	CPs showed better chondrogenesis
17	Fellows et al., 2017 16	OA human knee joints	70 U/mL: 1 h	Type I 300U/mL: 3 h	DMEM + 10% FCS • 0.1 mM ascorbic acid • 0.5 mg/mL L-glucose • 10 mM HEPES • 1 mM sodium pyruvate • 2 mM L-glutamine • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 500 cells/mL-20 min; 12 days Expansion: Monoclonal CPs 3,000 cells/cm2 up to p9	Characterization of a divergent progenitor cell sub-populations	CP colony expansion Proliferative, senescence index Differentiation Telomere length analysis	N	CD90 CD105 CD166 Stro-1 NOTCH1 CD34 CD45	Adipogenic Osteogenic Chondrogenic	Two subsets present: (a) Early senescence, lower average telomere length with differential potential (b) Extended viability with regenerative potential
18	Levato et al., 2017 36	Equine metacarpo phalangeal joints	0.2%: 2 h	Type II 0.075%: 12 h	DMEM + 10% FCS • 0.2 mM ascorbic acid • bFGF (5 ng/mL)	FAA (10 µg/mL): 500 cells/cm2-20 min; >6 days Expansion: Polyclonal enriched CPs up to p3	Potential of CPs in hydrogel for cartilage regeneration and biofabrication	Biochemical, RT-PCR: markers of chondrogenesis and hypertrophy Neocartilage formation and mechanical properties	N	CD105 CD73 CD90 CD29 CD44 CD49d CD106 CD166 CD45	Adipogenic Osteogenic Chondrogenic	CPs better than chondrocytes: neocartilage formation Low COL X and high PRG4 Bioprint model: MSC + CP
19	Tao et al., 2018 15	Mice knee joint	Trypsin and mechanical dissociation: not specified		DMEM + 10% FCS	FAA (10 µg/mL-20 µg/mL) 4,000 cells/mL; 10 days	Does fibronectin activate CPs through integrin ɑ5β1 receptor	Cell proliferation and migration assay ɑ5β1receptor blockade Chondrogenic differentiation FACS RT-PCR: COL I, COL II In vivo early OA model	N	CD105 CD166 Fibronectin CD34 CD45	Chondrogenic	CPs at 20 µg/mL: highest proliferation with marker expression FN enhance cartilage repair through ɑ5β1pathway
20	Anderson et al., 2018 71	Nondiseased human femoral condyle	70 U/mL: 20 min	Type I 300 U/mL: 4 h	Low glucose DMEM + 10% FCS • 0.1 mM ascorbic acid • 10 mM HEPES • TGβ1 (1 ng/mL) • FGF2 (5 ng/mL) Atmospheric oxygen: 5% and 20%	FAA (10 µg/mL): 4,000 cells/mL-20 min; 5PD Expansion: Monoclonal CPs up to passage 2 (22-24 PD) Fibronectin coated transwell inserts	Effect of physioxia on scaffold free neocartilage constructs using CPs	Biochemical Biomechanical Molecular analyses: COL I, COL II, and COL X	N	—	Chondrogenic	Single progenitor cell able to generate neocartilage tissue phenotype under physioxia
21	He et al., 2018 72	Sprague-Dawley rats: 8 weeks	—	0.25% type II collagenase: 8 h	Low-glucose DMEM + 10% FCS	FAA (10 µg/mL): 4,000 cells/mL-20 min; polyclonal expanded 80% to 90% confluence	Link protein N-terminal peptide as a stimulator for proteoglycan and COL II	Proliferation Migration Chondrogenic differentiation of CPs	N	CD90 CD73 CD105 CD44 CD34 HLA-DR	Adipogenic Osteogenic Chondrogenic	LPP compared to TGFβ showed higher proliferation and chondrogenic markers
22	Vinod et al., 2018 49	Adult rabbit knee joint	0.2%	0.04% Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid 62 mg/mL • L-glutamine 2.5 mM/L	FAA (10 µg/mL): 4,000 cells/mL-20 min; 5PD Expansion: 25,000 cells/cm2 sub-confluence	Intraarticular injection of CPs (1 × 106) in sodium hyaluronate for treatment of high-grade OA	Population doubling FACS Scaffold viability Synovial fluid-S100A12 Histology: OARSI scores Differentiation studies	N	CD90 CD81 CD49e	Adipogenic Osteogenic	Short term (12 weeks) treated joints lower grade and S100A12 compared to controls (sodium hyaluronate) and 24 weeks
23	Kachroo et al., 2018 26	Human Nondiseased and OA knee	0.2%	0.04% Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid 62 mg/mL • L-glutamine 2.5 mM/L • 10 mM HEPES • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 700 cells/mL-20 min; 10-12 days Expansion: 10,000 cells/cm2 p0 vs. p5	Compare current channels between chondrocytes and CP from early and late cultures, non-diseased and OA joints Create electrophysiological profile for CPs	Patch clamp studies RT-PCR (channel profile) FACS	N	CD105 CD73 CD90 CD106 CD54 CD44 CD151 CD49e CD146 CD166 CD34 CD45	—	Similar current profile (voltage gated K+ channels) OA CPs more sensitive to time in culture and inflammatory process
24	Jayasuriya et al., 2019 34	Nondiseased human knee joint	2 mg/mL: 30 min	Type I 1 mg/mL: 8 h	DMEM + 10% FCS • 2.7 µM L-glucose • 0.1 mM ascorbic acid • 100 mM HEPES • 0.1 mM sodium pyruvate	FAA (10 µg/mL), cells/mL: not reported-20 min; 10 days Expansion: • Monoclonal CPs • Immortalized clonal cell lines up to 1 week	CPs better contender than BM-MSCs in re-integrating and bridging fibrocartilage meniscal tears	Conditioned media experiments IHC: CD166 FACS mRNA: markers of chondrogenesis and hypertrophy Differentiation analysis	N	CD90 CD54 CD166 CD106	Adipogenic Osteogenic Chondrogenic	CP cell lines: Fibrocartilage meniscal tear repairs (SDF-1/CXCR4 chemokine axis) Resistant to hypertrophic differentiation
25	Torgomyan et al., 2019 42	Juvenile bovine metacarpal phalangeal	70 U/mL: 1 h	300 U mL/1: 3 h	DMEM + 10% FCS • 0.1 mM ascorbic acid • 100 mM HEPES	FAA cells/mL: not reported; 14 days Expansion: Polyclonal CPs	1-34 PTH effect on CP differentiation and subchondral bone and articular cartilage regeneration	Chondrogenic/osteogenic differentiation Rat in vivo study: osteochondral defect healing	N	—	Chondrogenic Osteogenic	Continuous PTH administration modulated chondrogenic differentiation and promoted cartilage regeneration compared to intermittent exposure
26	Zhang et al., 2019 73	Knee joints: human and mouse	—	0.2% Type II: 8 h + 5% FCS	DMEM + 15%FCS	FAA cells/mL: not reported-20 min Expansion: P4	Core RNAs and signaling pathways in OA CPs	Differentially expressed RNAs Signaling pathways Proliferative ability IHC/IF/western blot PRDM16, NCAM1, N-cadherin, CPNE1, and CTGF	N	CD105 CD90 CD73 CD29 CD44 CD34 CD45	Adipogenic Osteogenic Chondrogenic	OA CPs showed decreased proliferation therefore cartilage degeneration Differentially expressed: miRNA-322, miRNA-503, miRNA-467a, G0s2 Signaling pathway: MAPK, Hippo, Wnt pathways
27	Xue et al., 2019 33	Swine	—	0.1% Type II: 6-8 h	Low-glucose DMEM + 10% FBS	FAA (10 µg/mL): 4,000 cells/mL-20 min; 7-14 days Expansion: 2 × 104 cells/cm 80% to 90% confluence	Superiority of either BM-MSC, chondrocytes and CP with PHBV scaffold in cartilage repair: nude mice implants	Proliferative ability Chondrogenic characteristics: GAG, total collagen IHC: COL II, Saf-O RT-PCR: ACAN, SOX9, COL II	N	—	Chondrogenic	Chondrocytes better than CPs, but CPs better than BM-MSC in chondrogenesis
28	Vinod et al., 2019 41	Human OA knee	0.2%	0.04% Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid 62 mg/mL • L-glutamine 2.5 mM/L • 10 mM HEPES • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 4,000 cells/mL-20 min; 10-12 days Expansion: 10,000 cells/cm2 p2	In vitro analysis of PRP as a biological scaffold for CP	Platelet quantification FACS Differentiation studies IF: COL II	N	CD105 CD73 CD90 CD49e CD44 CD54 CD151 CD34 CD45	Adipogenic Osteogenic Chondrogenic	PRP scaffold-maintained viability and favored trilineage differentiation PRP was chondro-inductive in controls (CPs devoid of differentiation)
29	Vinod et al., 2019 27	Human Nondiseased knee	0.2%	0.04% Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid 62 mg/ml • L-glutamine 2.5 mM/L • 10 mM HEPES • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 700 cells/mL-20 min; 10-12 days Expansion: 10,000 cells/cm2 p2	Chondrocytes and CPs co-cultures bettered monocultures in terms of chondrogenesis	Growth kinetics FACS RT-PCR: Agg, SOX9, COL II RUNX2, MMP-13, COL X Live dead assay Histology: Saf-O	N	CD105 CD73 CD90 CD106 CD54 CD44 CD151 CD29 CD49e CD146 CD166 CD34 CD45	—	Co-cultures did not better than monocultures
30	Vinod et al., 2019 44	Human Nondiseased knee	0.2%	0.04% Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid • L-glutamine • HEPES • TGβ2 (1 ng/ml) • FGF2 (5 ng/mL)	FAA (10 µg/mL): 2,000 cells/well 20 min; 10-12 days Expansion: 10,000 cells/cm2 up to p2 M-SPIO labelling: At 30% to 40% confluence 20 h incubation with 3 different iron oxide concentration: 12.75 µg/mL, 25.5 µg/mL, and 38.25 µg/mL of culture medium	Optimal concentration of M-SPIO for labelling CPs with minimal effect on cellular viability, surface marker expression and differentiation	FACS Cell viability: 7AAD Intracellular iron levels Differentiation studies	N	CD105 CD73 CD90 CD106 7AAD CD34 CD45	Adipogenic Osteogenic Chondrogenic	CPs incubated with 12.75 µg/mL of M-SPIO successfully labelled the cells P9
31	Vinod et al., 2019 24	Human OA knee	Not reported	Not reported	CPs: DMEM-F12 Glutamax + 10% FBS • Ascorbic acid • L-glutamine • HEPES • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL) Chondrocytes: DMEM-F12 Glutamax + 10% FBS • Ascorbic acid • L-glutamine • HEPES	CPs: FAA (10 µg/mL): 2,000 cells/well 20 min; 10-12 days Expansion: Up to to p0 Chondrocytes: Expansion: 10,000 cells/cm2 up to p0	Compare fresh chondrocytes, cultured chondrocytes and chondroprogenitors for a differentiating surface marker	FACS Differentiation studies IHC: CD49e	N	CD105 CD73 CD90 CD29 CD49e CD34 CD45	Adipogenic Osteogenic Chondrogenic	Significant difference in CD markers were seen between fresh chondrocytes to the other groups. Cultured chondrocytes and CPs did not show any difference between them.
32	Newberry et al., 2019 35	Nondiseased human	2 mg/mL: 30 min	Type I 1 mg/mL: 8 h	DMEM + 10% FCS • 2.7 µM L-glucose • 0.1 mM ascorbic acid • 100 mM HEPES • 0.1 mM sodium pyruvate	CPs: FAA (10 µg/mL), cells/mL: not reported-20 min; 10 days Expansion: Monoclonal CPs BM-MSCs cell line (pRetro-E2 SV40 construct)	Characterize the influence of SDF-1 on CP adhesion, migration and gene expression SDF-1 conditioned CPs stimulate reintegration of fibrocartilage meniscal breaks	FACS: CXCR4 MTT assay GAG assay Gene expression Tensile force testing	N	CXCR4	—	CPs migrate in response to SDF-1 (comparable to BM-MSC), without alteration in gene expression SDF-1 conditioned CPs migrated into injured areas and facilitated reintegration
33	Wang et al., 2020 46	CBA and MRL mice	—	0.2% collagenase II: 8h	DMEM + 10%FCS	FAA (10 µg/mL) 1,000 cells/cm2: 20 min Expansion: up to 10 days	Assess potential of extracellular vesicles secreted by MRL and CAB mice strains in terms of its effect on chondrocyte migration and healing in a surgical model of OA	FACS Extracellular vesicle uptake miRNA-seq data analysis Proliferation assay IHC: COL I, COL II and ACAN	N	CD29 CD44 CD90 CD34 CD45	—	MRL-extracellular vesicles showed superior effect on chondrocyte migration and healing of OA
34	Kachroo et al., 2020 25	Human OA knee joints	12 IU	100 IU type II collagenase	DMEM-F12 Glutamax + 10% FBS 10 mM HEPES	FC, CD49e+ and CD49e-ve cells subjected to FAA (10 µg/mL): 2,000 cells/mL-20 min Expansion: 12 days	Assess CD49e/CD29 acquisition in CD49e-ve sorted cells at multiple time points and compare adherent and nonadherent culture conditions	FACS: Sorting CFE	N	CD49e CD29	—	Both adherent and nonadherent cultures showed significant CD49e and CD29 acquisition suggestive of inherent phenotypic drift
35	Lim et al., 2020 39	Equine metacarpo phalangeal joints	0.2%: 2 h	Type II 0.075%: 12 h	DMEM + 10% FCS • 0.2 mM ascorbic acid • bFGF (5 ng/mL)	FAA (10 µg/mL): 500 cells/cm2-20 min; >6 days Expansion: Enriched CPs up to p3	Potential of CPs in tyramine and methacryloyl gelatin for chondral defect repair	Biochemical, RT-PCR: markers of chondrogenesis and hypertrophy Neocartilage formation and mechanical properties	N	—	—	Bio-ink hydrogels with photo initiators show potential in cartilage repair
36	Diloksumpan et al., 2020 38	Equine metacarpo phalangeal joints	0.2%: 2 h	Type II 0.075%: 12 h	DMEM + 10% FCS • 0.2 mM ascorbic acid • bFGF (5 ng/mL)	FAA (10 µg/mL): 500 cells/cm2-20 min; >6 days Expansion: Enriched CPs up to p3	Biofabricate ceramic and thermoplastic microfibers and embed MSCs and CPs and assess impact on cell-survival and tissue regeneration	Evaluation of bioactivity: day 7 Mechanical properties of osteochondral units Cartilage deposition stain: sGAG, Saf-O and COL I	N	—	—	Printable osteochondral plug showed promising chondrogenesis and osteogenesis and was mechanically reinforced by neosynthesized matrix
37	Kachroo et al., 2020 17	Human OA knee joints	12 IU	100 IU Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid • L-glutamine • HEPES • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	CPs: FAA (10 µg/mL): 2,000 cells/well 20 min; 12 days Expansion: Up to to p4	Compare human platelet lysate as an xeno-free alternative for fetal bovine serum for expansion of CPs	Growth kinetics FACS RT-PCR: markers of chondrogenesis and hypertrophy Trilineage differentiation	N	CD105 CD73 CD90 CD29 CD49e CD146 CD166 CD34 CD45 HLA-DR	Adipogenic Osteogenic Chondrogenic	hPL served as an optimal alternative with higher alizarin uptake. It showed lower values of COL1, COLX, ACAN. and COLII.
38	Vinod et al., 2020 14	Human OA knee joints	12 IU	100 IU Type II collagenase	DMEM-F12 Glutamax + 10% FBS • Ascorbic acid • L-glutamine • HEPES • TGβ2 (1 ng/mL) • FGF2 (5 ng/mL)	CPs: FAA (10 µg/mL): 2,000 cells/well 20 min; 12 days Expansion: Up to to p3	Compare laminin and FAA derived CPs	Growth kinetics FACS RT-PCR: markers of chondrogenesis and hypertrophy Trilineage differentiation	N	CD105 CD73 CD90 CD29 CD49e CD146 CD166 CD34 CD45 HLA-DR	Adipogenic Osteogenic Chondrogenic	Laminin CPs show higher proliferative potential, higher osteogenic and adipogenic potential and lower COLII expression when compared to fibronectin assay derived CPS
39	Jessop et al., 2020 37	Human nasoseptal cartilage	2 mg/mL pronase	2.4 mg/mL collagenase: 16-16 h	DMEM/F12 + 10% FCS • 0.1 mM ascorbate-2-phosphate • 0.5 mg/mL L-glucose • 2 mM L-glutamine • 10 mM HEPES • TGβ1 (1 ng/mL) • FGF2 (5 ng/mL)	1. Chondrocytes, 2. Chondroprogenitors for FAA (10 µg/mL): 2000 cells/mL-20 min Expansion: Up to passage 13 3. Non-fibronectin adherent chondrocytes	Compare chondrogenic potential of CPs in comparison to chondrocytes and fibronectin depleted chondrocytes	Growth kinetics FACS Karyotyping RT-PCR Trilineage differentiation Histology	N	CD29 CD44 CD56 CD73 CD90 CD34 CD45	Adipogenic Osteogenic Chondrogenic	Chondrocytes expressed highest levels of COL II and BMP-2 with efficient Alcian blue uptake Mixed population of chondrocytes and CPs essential for optimal chondrogenesis. Cross talk via SDF-1 signaling
40	Morgan et al., 2020 43	Bovine metacarpo phalangeal joints	70 U/mL: 2 h	300 CDU/mL: 16 h	DMEM/F12 + 10% FCS • 50 µg/mL ascorbic acid-2mono-phosphate • 1g/L-glucose • 2 mM L-Glutamine • 10 mM HEPES • 1 mM sodium pyruvate	FAA (10 µg/mL): 1,000 cells/well-20 min; 6 days Expansion: Monoclonal: 22-27 PD	Screen optimal chondrogenic medium for equine CPs against a panel of known chondrogenic factors	sGAG quantification Toluidine blue: pellet RT-PCR: COL II, COL I, ACAN. SOX9	N	—	—	BMP-9 showed highest chondrogenic and morphogenic potential as an additive for chondrogenic differentiation of CPs
41	Mancini et al., 2020 40	Equine metacarpo phalangeal joints	0.2%: 2 h	Type II 0.075%: 12 h	DMEM + 10% FCS • 0.2 mM ascorbic acid • bFGF (5 ng/mL) • 4.5 g/L • 1% MEM-NEAA	FAA (10 µg/mL): 500 cells/cm2-20 min; >6 days Expansion: p4	Performance of multicomposite implant using CPs and MSCs (zonally) for the repair of osteochondral defect in ponies	sGAG (DMMB) IHC: COL I, COL II, ACAN Arthroscopy Histology scoring	N	—	—	Limited repair, persistence of hydrogel, zonal construct displayed higher stiffness
42	Kachroo et al., 2020 32	Human OA knee joint	12 IU	100 IU: overnight	DMEM-F12 Glutamax + 10% FBS	1. Freshly isolated cells: FC 2. CPs: FAA+ FAA (10 µg/mL): 2,000 cells/cm2 20 min; 12 days 3. Non-fibronectin adherent chondrocytes: FAA-ve	Assess potential of fibronectin in isolating cells and if they displayed any distinction from chondrocytes	RT-PCR: SOX9 ACAN COL IIA1 COL IA1 RUNX2 COL XA1	N	—	—	FAA+ve CPs showed significantly lower levels of RUNX2 and COLX when compared to FAA-ve group
43	Dai et al., 2020 47	Rat articular cartilage	0.2%	—	DMEM-F12 + 10% FCS	FAA (10 µg/mL) 4,000 cells/mL: 20 min-40 min Expansion: Early CP: p0 Late CP: p10	Can senolytic drugs improve outcome of joint arthroplasty by elimination senescent CPs and accelerating intermittent hydrostatic pressure induced chondrogenesis?	Cell proliferation Migration assay Differentiation In vitro/in vivo senolytic treatment: dasatinib and quercetin Micro-computed tomography Histological scoring: Saf-0, COL II	N	CD105 p53	Adipogenic Osteogenic Chondrogenic	Senolytic eliminated senescent CPs and decreased senescence associated secretory phenotype and improved joint distraction arthroplasty outcomes

FAA = fibronectin adhesion assay; OA = osteoarthritis; CP = chondroprogenitor; MSC = multipotent mesenchymal stromal cells/mesenchymal stem cells; BM-MSC = bone marrow mesenchymal stem cell; FBS = fetal bovine serum; hPL = human platelet lysate; CDU = collagen digestion units; IU = international unit; DMEM/F12 = Dulbecco’s minimum essential medium/F-12 Ham; AA = ascorbic acid; DMSO = dimethyl sulfoxide; NEAA = Non-essential amino acid; FACS = fluorescence assisted cell sorting; p = passage; PD = population doubling; HEPES = (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid); CD = cluster of differentiation; CFE = colony forming efficiency; NOTCH1 = notch homolog 1; TGFβ = transforming growth factor beta; FGF = fibroblast growth factor; IHC = immunohistochemistry; GAG = glycosaminoglycan; Saf-O = safranin O; RT-PCR = real-time polymerase chain reaction; COL I = type I collagen; COL II = type II collagen; ACAN = aggrecan; COL X = type X collagen; SOX9 = SRY box transcription factor 9; RUNX2 = runt related transcription factor-2; MMP = metalloproteinase; PRG4 = proteoglycan 4; SDF = stromal derived factor; CXCR4 = C-X-C chemokine receptor 4; BMP = bone morphogenic protein; 7AAD = 7 aminoactinomycin D; MTT = (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide); SEM = scanning electron microscopy; TEM = transmission electron microscopy; SCID = severe combined immunodeficiency; LPP = link protein N-terminal peptide; PTH = parathormone; PRP = platelet rich plasma; S100A12 = S100 calcium binding protein; PRDM16 = PR domain containing 16; NCAM1 = neural cell adhesion molecule 1; CPNE1 = copine 1; CTGF = connective tissue growth factor; Wnt = wingless related integration site; MRL = Murphy Roths Large; DMMB = dimethyl methylene blue; SPIO = superparamagnetic iron oxide; MAPK = microtubule associated protein kinase; PHBV = polyhydroxy butyrate co-hydroxy valerate.

Isolation Protocols

For tissue digestion and cellular dissociation, varying durations and enzymatic concentrations were reported. Types of enzymes employed for cartilage digestion included collagenase I (n = 13), collagenase II (n = 18), collagenase (type unspecified, n = 6), a combination of collagenase I, II, and dispase (n = 1), and trypsin (with mechanical dissociation, n = 1). From the data analyzed, most studies (n = 38), reported the use of pronase followed by collagenase digestion of cartilage tissue for cell isolation. Since cellular dissociation and expansion in culture can cause a change in cellular phenotype, the collated data were further analyzed for specific culture conditions such as medium used, seeding density, and additives/growth factors utilized for monolayer expansion. ¹³

Culture Medium/Additives

All the studies used either DMEM (Dulbecco’s minimum essential medium) or DMEM-F12 as the basal medium, supplemented with either fetal bovine serum (FBS; 10%, n = 38; 15%, n = 1; 40%, n = 3) or human platelet lysate (10%, n = 1). Except for 7 studies, all others reported the use of additional supplements and growth factors. The additives included were ascorbic acid (0.0028 mM to 28 mM, n = 23), L-glutamine (2-2.5 mM, n = 15), sodium pyruvate (0.1-1 mM, n = 6), and HEPES (10-100 mM, n = 19). With respect to growth factors, most commonly used additions were of either transforming growth factor beta 1 (TGFβ1, 1 ng/mL, n = 3), fibroblast growth factor 2 (FGF2, 5 ng/mL, n = 5), or combinations such as TGFβ1 + FGF2 (n = 5) and TGFβ2 + FGF2 (n = 8). Other unique modifications to the culture medium, intended to maintain chondrogenic phenotype included, use of low glucose (0.0027-2.78 mM, n = 14) and hypoxic conditions (5% O2, n = 1).

Seeding Density

Fibronectin adhesion assay protocol described by Dowthwaite et al. was commonly followed in all the published studies with a few variations. ⁷ All the reported studies involved fibronectin coating at a concentration of 10 µg/mL (n = 41). Additionally, one study compared fibronectin assay–derived chondroprogenitors to laminin adhesion assay (10 µg/mL), ¹⁴ while another study compared the standard protocol to a higher fibronectin concentration (20 µg/mL), which yielded cells displaying higher proliferation and marker expression. ¹⁵ Seeding densities mentioned for FAA varied between 1,000 and 4,000 cells/well (n = 32) or 700 cells/mL (n = 3) or 500 cells/cm² (n = 5), for a period of 20 minutes (n = 40), following which the nonadherent cells were removed. The adherent cells were further expanded for 6 to 12 days, ensuring 5 population doublings. Clonally expanded progenitors were grown as polyclonal enriched cultures (n = 23) or as monoclonal cultures (n = 6). A report also exhibited the ability of progenitors to expand in culture up to passage 9 while retaining good viability and regenerative potential. ¹⁶

Characteristics of Chondroprogenitors

Growth Kinetics

With regard to growth kinetics, in the two studies that compared chondroprogenitors to chondrocytes, one reported higher colony-forming efficiency and 2.5-fold greater telomerase activity in the former, along with exponential growth recorded up to 20 population doublings, ⁹ while the other demonstrated comparable results between the two cell types. ²⁴ Characterization by Fellows et al. demonstrated the presence of two progenitor subsets, one exhibiting early senescence and lower average telomere length with higher differentiation potential while the other showed extended viability with greater regenerative potential. ¹⁶ In another study, chondroprogenitors isolated using a laminin adhesion assay showed significantly higher proliferative potential but lower chondrogenic potential. ¹⁴ It was also seen that for creation of xeno-free culture conditions, when human platelet lysate was used in place of FBS, progenitors displayed greater proliferation with lower expression of type I and X collagen (fibrocartilage and hypertrophy markers). ¹⁷ With regard to culture conditions producing maximum growth of progenitors, Melero et al. report an optimal seeding density of 10,000 cells/cm² and medium volume of 0.3 mL/cm².^18,19 The authors further demonstrated an efficient post-cryoviability and 17-fold expansion potential using macroporous microcarrier supplemented with 40% FCS and 1 ng/mL of TGFβ1. ²⁰

Surface Markers

Multiple reports examined chondroprogenitors for a gamut of cell surface markers, investigating positive, negative MSC marker expression and differentiation potential to assess conformation to the ISCT criteria. Of the 41 reports, 25 studies assessed cell surface marker expression, while 30 evaluated potential for differentiation (either monolineage or trilineage) and none of the studies reported evaluation based on the complete minimum criteria laid down for MSCs by ISCT 2006. Regarding differentiation potential, it was seen that intermittent hydrostatic pressure (n = 1) ²¹ and growth under physioxia (n = 1), ²² demonstrated better chondrogenesis. In another study, higher adipogenesis and osteogenesis was observed in chondroprogenitors derived from laminin adhesion compared to fibronectin adhesion. ¹⁴ Supplementation with platelet lysate as opposed to FBS caused chondroprogenitors to show comparatively higher alizarin red uptake. ¹⁷ Commonly reported positive MSC markers included CD105 (n = 17), CD90 (n = 20), CD73 (n = 12), while negative MSC markers included CD34 (n = 15) and CD45 (n = 15). Numerous reports evaluated the following cell surface proteins as potential markers of chondrogenesis namely CD29 (n = 12) in combination with CD49e (n = 19), CD146 (n = 4), and CD166 (n = 13). Fibronectin is a classical ligand for the α5β1 integrin receptor, for which CD49e is the α5 component. ²³ CD49e was shown to be highly expressed by superficial layer chondroprogenitors when compared to freshly isolated chondrocytes. However, when expression was compared to cultured chondrocytes, no significant difference was seen between the 2 groups. ²⁴ In another report, when CD49e negative cells were assessed in monolayer and suspension culture systems, dramatic upregulation of CD49e expression was noted. ²⁵ Similarly, with regard to CD146 and CD166, no consensus has been found to suggest that sorting based on aforementioned markers will yield cells with enhanced chondrogenic potential.^26,27

Chondrogenic Potential: Chondroprogenitors versus Other Cell Types

Initial characterization of superficial layer chondroprogenitors revealed expression of notch receptors, a gene known to be involved in cellular colony-forming ability.^7,28 Later these receptors were also confirmed to be present in progenitors embedded at deeper cartilage zones. ²⁹ With respect to chondrogenic potential, Xue et al. reported the superiority of articular cartilage derived chondroprogenitors to progenitors isolated from other sources such as auricle and intervertebral disc. ³⁰ However, a study employing human nasal septum–derived chondroprogenitors reported better results when compared to articular cartilage–derived chondroprogenitors, adipose tissue–derived MSCs, and BM-MSCs. ³¹ In addition to this, many studies exhibited superiority of chondroprogenitors to BM-MSCs as a potential candidate for cartilage repair. Literature by McCarthy et al. and Kachroo et al. demonstrated significantly low levels of hypertrophy markers in chondroprogenitors,^1,32 while Xue et al. reported higher expression of chondrogenic genes when compared to BM-MSCs.^30,33 Similarly, Jayasuriya et al. and Newberry et al. showed that chondroprogenitors bettered BM-MSCs for meniscal tear repairs, demonstrating higher migratory potential in response to SDF-1/CXCR4 (SDF, stromal derived factor; CXCR4, C-X-C chemokine receptor 4) with improved resistance to hypertrophy.^34,35 On the same line, Khan et al. and Levato et al. reported superiority of chondroprogenitors to chondrocytes, due to higher SOX9, lubricin (PRG4) levels, and lower type X collagen expression, thus indicating improved potential for neocartilage formation.^8,36 Moreover, Vinod et al. demonstrated comparable results in an in vitro human study, ²⁷ while Jessop et al. and Xue et al. reported chondrocyte superiority with regard to type II collagen expression and glycosaminoglycan production.^33,37

Scaffold Use, Specific Additives, and In Vivo Studies for Cartilage Repair

Scaffold-based studies (n = 9) using chondroprogenitors showed variable results ( Table 1 ). An in vitro study employing a bio-fabricate made of ceramic and thermoplastic microfibers, further embedded with chondroprogenitors and BM-MSCs exhibited favorable neosynthesized matrix upon mechanical stimulation. ³⁸ Similarly, Lim et al. reported chondroprogenitors embedding in light visible bioink hydrogel made up of tyramine and methacryloyl gelatin and displayed potential for integration and intraoperative bioprinting. ³⁹ However, in an in vivo assessment performed by Mancini et al., cell-laden multicomposite implants showed limited repair and persistence of the hydrogel with zonal construct displaying higher stiffness. ⁴⁰ In a report by Vinod et al., platelet-rich plasma (PRP), a biological scaffold was used and resulted in favorable glycosaminoglycan deposition even in the absence of chondro-induction. ⁴¹ With regard to the effect of specific supplements, added in order to enhance chondroprogenitor phenotype, three different studies were reported. Torgomyon showed that continuous (rather than intermittent) administration of parathormone (PTH) modulated chondrogenic differentiation and enhanced cartilage regeneration in an in vivo osteochondral defect model. ⁴² In a study by Jayasuriya et al., SDF1 conditioned chondroprogenitors displayed, higher migratory potential into areas of injury with promotion of regeneration, in an in vitro meniscal defect model. ³⁴ Additionally, Morgan et al. showed that addition of bone morphogenic protein (BMP-9, 100 ng/mL) to the chondrogenic differentiation medium, displayed the highest chondrogenic and morphogenic potential when compared against a panel of known chondrogenic factors. ⁴³

In the literature search, only one study by Vinod et al. reported on the successful labelling of chondroprogenitors with 12.75 µg/mL of micron-size superparamagnetic iron-oxide that had minimal effect on viability, surface marker expression, and differentiation potential. ⁴⁴

The first in vivo study to use chondroprogenitors was a pilot by Williams et al., performed utilizing a caprine chondral defect model. ⁹ It reported excellent integration, although no difference in the quality of repair tissue was observed when compared to full-depth chondrocytes. In another equine study employing a chondral defect model, autologous chondroprogenitors embedded in a fibrin scaffold displayed superior repair to allogeneic chondroprogenitors and also to the scaffold itself. ⁴⁵ A study performed by Wang et al. demonstrated the use of intraarticular injections of chondroprogenitor extracellular vesicles secreted by MRL mice, showing attenuation of osteoarthritis, thus providing new insight in their role as exosomal injections. ⁴⁶ Additionally, another study by Dai et al. reported novel use of Dasatinib and Quercetin in eliminating senescent progenitors, thereby decreasing senescence-associated secretory phenotype, thus improving joint distraction arthroplasty outcomes. ⁴⁷ The literature search also revealed that a few studies involving fibronectin adhesion–derived chondroprogenitors exhibited variable results. One study which utilized labelled chondroprogenitors, implanted into severe combined immunodeficiency (SCID) mouse thigh muscle, showed maintenance of chondrogenic potential but failure to produce extracellular matrix (ECM). ⁴⁸ Similarly, another study employing polyhydroxy butyrate co hydroxy valerate (PHBV)-chondroprogenitor constructs as subcutaneous implants demonstrated better chondrogenesis than BM-MSCs but inferior results to chondrocytes. ³³ Moreover, another study revealed that chondroprogenitors embedded with hyaluronic acid also failed to show superiority to plain hyaluronic acid when injected intraarticularly in a rabbit-based monosodium iodoacetate–induced high-grade osteoarthritic model. ⁴⁹ Another study by Mancini et al. using a long-term equine model reported limited production of cartilage-like tissue in osteochondral defects when implanted with a 3D multicomposite construct containing chondroprogenitors and MSCs. ⁴⁰

Migratory Chondroprogenitors

A total of 18 reports describing chondroprogenitors isolated from cartilage explants by virtue of their migratory properties were published ( Table 2 ).

Table 2.

Summary of Cartilage Explant–Derived Migratory Chondroprogenitors from Articular Hyaline Cartilage.

S. No.	Study reference	Cartilage: Species	Isolation Culture Condition			Objectives	Outcome Measures	Results
			Cartilage Slices: Dimensions	Culture Medium and Additives	Time for Cellular Dissociation/Migration			ISCT Criteria	Reported CD Markers	Differentiation Potential	Overall
1	Koelling et al., 2009 10	Human OA knee joints	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM	Outgrown cells after 10 days expanded at 1,000 cells/cm2	Identify CP population in severe osteoarthiritic cartilage	FACS Trilineage RTPCR: SOX9, ACAN, COL II, COL I, and RUNX2 IHC: STRO-1 ISH: COL II Migration assay Microarray	N	CD13, CD14, CD18, CD24, CD29, CD31, CD34, CD36, CD44, CD45, CD73, CD90, CD105, CD106, CD117, CD166, CD271, α2-α6, β1, STRO-1	Adipogenic Ostoegenic Chondrogenic	CP show high migratory and chondrogenic potential; knockdown of RUNX2 caused upregulation of SOX9 and enhanced
2	Koelling et al., 2010 60	Human OA knee joints	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM	Outgrown cells after 10 days expanded at 1,000 cells/cm2	Effect of sex-specific differences and sex steroids on CP in late stage OA	FACS RTPCR: SOX9, COL II, COL I, and RUNX2 IHC and western blot: Estrogen receptor α and β, androgen Microarray	N	Estrogen and androgen receptors	—	Correlation between sex steroid levels and gene expression regulation of SOX9, collagen, and RUNX2
3	Seol et al., 2012 51	Bovine tibial plateau and human osteochondral explants: Talus	25 mm2	DMEM-F12 + 10% FBS L-Ascorbate 50 µg/mL	Explant culture equilibration: 2 day X shape matrix tear: 0.5 mm depth followed by 5- to 7-day culture Trypsin-EDTA 0.25%: 10 minutes	CPs migrate and respond to cartilage injury	Live dead assay IF: PCNA Cell migration/chemotaxis assay Trilineage differentiation Microarray: ABCG2, CDFLK-1, RUNX2, SOX9	N	CD73, CD90, CD105, CD39, CD29	Adipogenic Osteogenic Chondrogenic	Injury induces homing of CPs via HMGB-1 and RAGE mediated chemotaxis
4	Joos et al., 2013 53	Non-fibrillated human OA knee joints	4 mm	DMEM + 10% FBS L-glutamine 2 mM	Outgrown cells after 14 days	Influence of growth factors and cytokines on CP migration	ICC FACS Chemotaxis Attachment and migration assay Supernatant: cytokine	N	CD90, CD54, CD166, CD73, Actin, Vinculin, CD44, CD146, CD133, MSCA-1, STRO-1	Adipogenic Osteogenic Chondrogenic	Traumatized cartilage releases chemoattractant like PDGF-BB and IGF-1; cytokines like IL-1β and TNFα inhibit CP migration
5	Zhou et al., 2014 58	Bovine tibial plateau	25 mm2	DMEM-F12 + 10% FBS L-Ascorbate 50 µg/mL	Explant culture equilibration: 2 days X shape matrix tear: 0.5 mm depth followed by 7- to 10-day culture Trypsin-EDTA 0.25%: 10 minutes Expansion: not mentioned	Comparative analysis between CP, chondrocytes, synoviocytes and synovial fluid cells	Microarray RT-PCR: 1L-6, 1L-8, CCL2, CXCL12, SOX9, RUNX2 and matrix forming gene expression sGAG assay	N	—	—	CPs overexpressed IL-8, CCL2 and were phenotypically similar to synoviocytes than chondrocytes
6	Matta et al., 2015 56	Human OA explant adjacent to main defect	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM	Outgrown cells after 10 days Expansion: 100 cells/cm2 immortalized by lentiviral (hTERT) transfection	Understanding Ca2+ homeostasis in CP	RT-PCR: SOX9, COLIIA1, Aggrecan, RUNX2, COL1A1, COL10A1, and molecules involved in Ca2+ homeostasis Western blot: calcium homeostasis molecules IF: Ca2+ measurement: Fura-2	N	—	—	CPs express IP3R, STIMI and Orai1 proteins and negative for RYR, possible role of Ca2+ signaling in differentiation
7	Jang et al., 2014 55	Bovine stifle joints	2.5 cm2	45% DMEM + 45% HAM-12 + 10% FBS	Explant culture equilibration: 2 days Blunt injury followed by 5- to 7-day culture Trypsin-EDTA: 10 minutes	Low-intensity pulsed ultrasound stimulated (LIPUS) migration of CP to injured area	Western blot: Focal adhesion kinase (FAK) Cell migration assay: confocal	N	—	—	LIPUS (5 minutes; 27.5 mW/cm2 at 3.5 MHz) favored migration of CP through FAK pathway
8	Elsaeaaer et al., 2016 52	Human nasal septum 52	1 mm2	DMEM-F12 + 10% FBS	Minced cartilage: 7 days for outgrowth culture Expanded to sub confluence	Characterization of adult nasal CPs against chondrocytes and BMMSC	Population doubling FACS Migration assay and effect of chemotactic factors RT-PCR: COL I, COL II, ACAN, and FABP4 Chondrogenic induction: decellularized ECM	N	CD34, CD45, CD133, CD31, CD9, CD29, CD44, CD49e, CD49f, CD54, CD73, CD90, CD105, CD106, CD146, CD166	Adipogenic Osteogenic Chondrogenic	CP had higher migratory capacity compared to BMMSC and chondrocytes Showed similar ECM secretion to chondrocytes CP showed higher CD106 and CD90 to MSC, lower CD9 to chondrocyte
9	Zhou et al., 2016 59	Nondiseased bovine tibial plateau	25 mm2	DMEM-F12 + 10% FBS L-Ascorbate 50 µg/mL	Explant culture equilibration: 2 day X shape matrix tear: 0.5 mm depth followed by 5- to 7-day culture Trypsin-EDTA 0.25%: 10 minutes	Compare inherent phagocytic activity of CPs in comparison to chondrocytes	Pulse chase experiment Lysosome activity: fluorescent probes RT-PCR and western blot: phagocytosis related genes and proteins	N	—	—	CPs internalized more debris, higher lysosome activity and mass and revealed time and cathepsin B dependent clearance of cell debris compared to chondrocytes
10	Wang et al., 2017 63	Bovine tibial plateau: normal joint	25 mm2	DMEM-F12 + 10% FBS L-Ascorbate 50 µg/mL	Explant culture equilibration: 2 days X shape matrix tear: 0.5 mm depth followed by 7-day culture Trypsin-EDTA 0.25%: 10 minutes Expansion: 28,000 cells/cm2	Determine potential of CP to stimulate VEGF expression	IHC, RT-PCR, and Western blot for VEGF SDF 1α antagonist and MAPK inhibitors for pathway linking of SDF 1 and VEGF	N	—	—	CP stimulate VEGF expression by SDF 1α secretion dependent on p38 MAPK activation
11	Batschkus et al., 2017 61	Human OA knee joint 61	—	DMEM Glutamax + 10% FBS	Explant culture: 10 days Migrated cells immortalized: lentiviral transfection (hTERT)	Analysis of secretome of undifferentiated and differentiated CPs	Liquid chromatography-coupled tandem mass spectrometry	N	—	—	CPs produce chondrocyte like tissue ex vivo, CP differentiation in alginate enhances chondrogenic potential
12	Schminke et al., 2016 64	Human RA and OA knee joint	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM	Explant culture: 10 days Outgrown cells expanded at 1,000 cells/cm2	Assess effect of CPs with IL-17 receptor positivity on chondrogenic potential	FACS RT-PCR: SOX9, COL2A1, RUNX2, COL1A1, MMP13, TIMP1, TIMP3, and IL-6 IHC: IL-17 Cell differentiation and migration assay	N	CD29, CD73, CD44, CD45, CD34, CD105, CD90	Adipogenic Osteogenic Chondrogenic	IL-17 reduced chondrogenic potential by upregulation of RUNX2, enhanced IL-6 and MMP13
13	Nguyen et al., 2018 50	Human femoral condyle and knee joint 50	8-15 mm3	Coon’s-F12 + 10% FBS or 5%PL L-glutamine 2 mM/L	Expansion: 12 months	Potential of PL on CPs and differentiated chondrocytes	Cell proliferative assays Proliferation/survival pathways IHC: COL I, COL II, COL X, Cyclin D1	N	—	—	PL upregulated proliferation and survival of CPs (ERK, Akt and CyclinD1) and induced HIF-1 alpha increase PL induced release of CPs from explants as compared to control
14	Janssen et al., 2019 62	Human cartilage: lateral condyle: adjacent to defect area 62	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM/L	Outgrown cells after 10 days expanded at 1000 cells/cm2	Influence of TGF-β3, EGF, and biglycan on SOX9 and RUNX2 expression in CPs, diseased chondrocytes, and immortalized CPs	RT-PCR/IHC/immunoblotting: chondrogenic and osteogenic markers, TGFβR1, TGFβ3	N	—	—	TGFβ3 and EGF stimulation influenced biglycan, SOX9 and RUNX2 TGFβR1 and EGFR Contribute to degenerative/regenerative changes of late OA
15	Wagner et al., 2019 74	Human cartilage: lateral condyle: adjacent to defect area 74	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM/L	Outgrown cells after 10 days expanded at 1000 cells/cm2	Chemoattractant role of High Mobility Group Box 1 Protein on OA knee tissue and CPs	Immunoblotting/IHC/in situ hybridization: HMGB1	N	—	—	HMBG1 release by chondrocytes has a migratory effect on CPs mediated by RAGE and TLR4 in vitro
16	Wang et al., 2019 65	Nondiseased human knee joints	10 mm3	DMEM-F12 + 10% FBS	Outgrown cells after 14 days expanded to p3	Compare effect on PRP on CPs, MSC, and chondrocytes in a chondral defect rabbit model	Growth kinetics CCK-8 assay Histology: alcian blue, toluidine blue, Saf O IHC: COL II RT-PCR: ABCG2, Notch 1, RUNX2, PPARγ, COL I, COL II, SOX9, ACAN	N	CD29, CD31, CD45, CD90, CD105, STRO-1, Platelet associated surface markers: CD41a, CD42b	Adipogenic Osteogenic Chondrogenic	CPs displayed superiority over BM-MSC, chondrocytes and PRP alone in PRP mediated chondral defect regeneration
17	Matta et al., 2019 57	Human OA knee joints	8-15 mm3	DMEM-F12 + 10% FBS L-glutamine 10 mM	Outgrown cells after 10 days expanded at 1000 cells/cm2	To explore surfaceome and define biomarkers of human knee OA articular cartilage derived CPs	Biotinylation Glycocapture Shotgun proteomics	N	—	—	Specific to CPs: Transporters: TRPM2, KCNJ2, KCNQ2 Receptors: PTPRB, PTPRM and Syntaxin-4 Enzymes: ALK tyrosine kinase receptor, carbonic anhydrase 12 ECM proteins: Syndecan 3, agrin, filaggrin Upregulation of Integrin α2
18	Wang et al., 2020 54	Human OA knee joints	0.1 mm3 digested with 0.1% collagenase type II: 2 hours	DMEM-F12 + 10% FBS	Outgrown cells after 10 days expanded to p3	Compare grade 1-2 vs. grade 3-4 isolated CPs in terms of immunophenotype, differentiation potential and migratory capacities	Growth kinetics CCK-8 assay CFU-F assay FACS Differentiation potential RT-PCR: RUNX2, OCN, CEBP/α, PPARγ, Col II, SOX9 RNA Seq analysis	N	CD29, CD31, CD44, CD45, CD73, CD90, CD105, CD166, CD271	Adipogenic Osteogenic Chondrogenic	CPs derived from grade 3-4 were dysfunctional and displayed higher levels of CD105 and CD271, enhanced osteo-adipogenic potential and decreased chondrogenic potential

OA = osteoarthritis; RA = rheumatoid arthritis; CP = chondroprogenitor; MSC = multipotent mesenchymal stromal cells/mesenchymal stem cells; BM-MSC = bone marrow mesenchymal stem cell; FBS = fetal bovine serum; IU = international unit; DMEM/F12 = Dulbecco’s minimum essential medium/F-12 Ham; AA = ascorbic acid; FACS = fluorescence assisted cell sorting; p = passage; CD = cluster of differentiation; TGFβ = transforming growth factor beta; PRP = platelet rich plasma; ICC = immunocytochemistry; IHC = immunohistochemistry; ISH = in situ hybridization; GAG = glycosaminoglycan; Saf-O = safranin O; COL I = type I collagen; COL II = type II collagen; ACAN = aggrecan; COL X = type X collagen; SOX9 = SRY box transcription factor 9; RUNX2 = runt related transcription factor-2; PCNA = proliferating cell nuclear antigen; SDF = stromal derived factor; HMGB = high motility group box protein-1; PPARγ = peroxisome proliferator-activated receptor gamma; ABC = ATP-binding cassette transporters; PDGF = platelet derived growth factor; IGF-1 = insulin like growth factor; IL = interleukin; TNF = tumor necrosis factor; MSCA = mesenchymal stem cell antigen; CCL2 = chemokine ligand 2; CXCL = chemokine CC motif ligand; FABP4 = fatty acid binding protein; ECM = extracellular matrix; VEGF = vascular endothelium growth factor; MAPK = mitogen activated protein kinase; TIMP = tissue inhibitor of metalloproteinases; PL = platelet lysate; ERK = extracellular-signal-regulated kinase; Akt = serine/threonine kinase; HIF-1 = hypoxia inducible factor-1; ECF = anti epidermal growth factor; ECFR = anti epidermal growth factor receptor; RAGE = receptor for advanced glycation end-products; TLR4 = Toll like receptor-4; TRPM2 = transient receptor potential cation channel; KCN = inward rectifying potassium channel; PTPRB = receptor-type tyrosine-protein phosphatase beta; PTPRM = receptor-type tyrosine-protein phosphatase mu; ALK = anaplastic lymphoma kinase tyrosine kinase receptor; OCN = osteocalcin; CEBP = CCAAT-enhancer-binding proteins; CCK = cell counting kit; CFU-F = colony-forming unit-fibroblast; RT PCR = real-time polymerase chain reaction; EGFR = epidermal growth factor receptor.

Source of Tissue

Studies involving human subjects (n = 13), utilized hyaline cartilage either sourced from articular areas (n = 12) or the nasal septum (n = 1). Human samples were derived from diseased (osteoarthritis: n = 13, rheumatoid arthritis: n = 1) or nondiseased (n = 1) joints. Five studies used healthy bovine articular cartilage for the isolation of migratory chondroprogenitors.

Cell Isolation and Culture Conditions

To isolate migratory chondroprogenitors, cartilage slices of varying sizes were obtained and cultured as explants for 7 to 14 days. With respect to the size of cartilage explants, most sliced specimens had a size of 8 to 15 mm² (n = 8), while others were 25 mm² (n = 6), 10 mm³ (n = 1), 4 mm (n = 1), 1 mm² (n = 1), 0.1 mm³ (n = 1), and unspecified (n = 1). In a few studies (n = 4), following 2 days of culture, explants were scratched to create a matrix tear (X-shaped) of 0.5 mm depth, and further cultured for 7 days, following which they were treated with 0.25% Trypsin-EDTA (10 minutes) to facilitate easier release of migrated progenitors from the surface. Thereafter, outgrowing or migrated cells were reloaded at a seeding density of 1,000 cells/cm² and further expanded to confluence. All the studies used either DMEM (Dulbecco’s minimum essential medium) or DMEM-F12, further supplemented with FBS (10%, n = 18) or platelet lysate (5%, n = 1). Addition of ascorbic acid (0.28 mM, n = 4) and L-glutamine (2-10 mM, n = 9) was also reported. Use of additional growth factors for proliferation was not reported in any of the studies. Concerning proliferative capacity, one study reported the ability of progenitors to expand when cultured for a period of 12 months, maintaining its viability and inherent characteristics. ⁵⁰

Characteristics of Chondroprogenitors

Growth Kinetics: Migratory Potential

In 2009, Koelling et al. were the first to identify, characterize, and report migratory chondroprogenitors isolated from osteoarthritic cartilage. ¹⁰ They demonstrated presence of high migratory and chondrogenic potential. Additionally, Seol et al. showed that progenitors in response to cartilage injury displayed higher migration via High Mobility Group Box 1 Protein (HMGB) and receptor for Advanced Glycation End Products (RAGE) mediated chemotaxis. ⁵¹ Elsaesser et al. also reported superiority of nasal chondroprogenitors when compared to chondrocytes and BM-MSC with respect to migratory capacity. ⁵² A study by Joos et al. reported that release of platelet derived growth factor (PDGF-BB) and insulin-like growth factor (IGF-1) enhanced chondroprogenitor migration, whereas interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα) displayed an inhibitory influence on progenitor movement, following injury. ⁵³ Recently, Wagner et al. identified the vital role of chondrocytes in the release of HMGB-1 protein which influenced the migratory potential of chondroprogenitors via RAGE and toll like receptor 4 (TLR4). ⁵⁴ Another study reported the use of low-intensity pulsed ultrasound stimulated (LIPUS) migration of chondroprogenitors to the area of injury via focal adhesion kinase pathway. ⁵⁵

Surface Markers

From the literature analyzed, 7 reports investigated cell surface proteins to look for positive, negative MSC marker expression and differentiation potential. Commonly reported positive MSC markers included CD105 (n = 6), CD90 (n = 7), CD73 (n = 6), while negative markers reported were CD34 (n = 3) and CD45 (n = 5). Numerous reports also evaluated the following cell surface proteins as potential markers of chondrogenesis, namely, STRO-1 (n = 3), CD29 (n = 6), CD49e (n = 2), CD146 (n = 2), and CD166 (n = 4). With regard to trilineage differentiation, chondroprogenitors derived from grade 3 to 4 osteoarthritis (as opposed to grade 1-2) demonstrated higher dysfunction with greater osteo-adipogenic potential and decreased chondrogenic potential. ⁵⁴ With regard to cellular differentiation, a study reporting on proteins involved in Calcium signaling demonstrated chondroprogenitors to express IP₃, STIM1, and ORAI-1 receptors and absence of ryanodine receptors. ⁵⁶ A recent study by Matta et al. using biotinylation, glycocapture and shotgun proteomics, surfaceome of chondroprogenitors were analyzed in comparison to BM-MSCs. ⁵⁷ It was reported that chondroprogenitors demonstrate significantly higher levels of ECM proteins such as Syndecan, Agrin, and Filaggrin. They also reported notable upregulation of CD49b, an integrin marker and provided data pertaining to chondroprogenitor specific channels, including those belonging to the transient receptor potential melastatin (TRPM) family and potassium channel subfamily. ⁵⁷

Chondrogenic Potential: Chondroprogenitors versus Other Cell Types

Zhou et al. were the first to compare chondroprogenitors to chondrocytes, synoviocytes, and synovial fluid cells. ⁵⁸ It was seen that chondroprogenitors displayed more similarity to synoviocytes than chondrocytes, with overexpression of IL-8 and chemokine ligand-2 (CCL2). In other studies, progenitors showed higher phagocytosis and lysosomal activity dependent on cathepsin B mediated clearance ⁵⁹ but similar ECM production when compared to chondrocytes. ⁵²

Effects of Interventions

Koelling et al. reported a correlation between sex steroids and chondroprogenitors, that is, hormone replacement therapy provided in combination with physiological levels of testosterone demonstrated a positive influence on chondrogenic potential. ⁶⁰ In another study, human platelet lysate showed activation and proliferation of quiescent cells, thereby contributing to new cartilage formation. ⁵⁰ Additionally, differentiation of chondroprogenitors in alginate demonstrated favorable chondrogenesis. ⁶¹ When chondroprogenitors were stimulated with TGFβ3 and epidermal growth factor in vitro, upregulation of both SOX9 and runt related transcription factor-2 (RUNX2) levels was observed. ⁶² A few reports also described the variable influence of chondroprogenitors in a pathological setting. Wang et al. reported that chondroprogenitors might contribute to the pathogenic accumulation of vascular endothelial growth factor in an osteoarthritic environment, ⁶³ while another study found that chondroprogenitors isolated from rheumatoid arthritis joints displayed high levels of IL-17, thus contributing to reduced chondrogenic potential (via RUNX2, IL-6, and matrix metalloproteinase-13). ⁶⁴

In Vivo Studies for Cartilage Repair

Only one in vivo study involving chondroprogenitors derived from nondiseased human tissue was reported. ⁶⁵ In this rabbit chondral defect model by Wang et al., chondroprogenitors suspended in PRP showed higher chondrogenic predisposition with superior healing of critical defects when compared to BM-MSCs in PRP, chondrocytes in PRP, and PRP alone.

Chondroprogenitors Derived Following Cell Sorting

Following literature search, 4 studies were found to use cellular sorting, to isolate either single cells or obtain a subset based on cell surface marker expression ( Table 3 ). In a report by Su et al., human chondroprogenitors were isolated based on CD146 positivity, and when compared to unsorted chondrocytes, exhibited higher levels of MSC specific antigens and chondrogenic capacity in a 3-dimensional pellet culture. ¹¹ In another study by Yu et al., bovine chondrocytes were subjected to flow assisted single-cell sorting, seeded on gelatin-coated plates, and expanded in culture using 10% knock out serum replacement (KOSR). Following clonogenicity screening, cells with high-efficiency colonies were labelled as progenitors, wherein superficial layer derived cells displayed improved clonality, whereas deeper zone cells displayed higher chondrogenesis and osteogenesis. ¹² Furthermore, these cells were used in another study as a proof of concept, where a bioprinting model used progenitors to generate vessel-like channels. ⁶⁶ One report also assessed the effect of leptin on etiology and development of osteoarthritis using murine progenitors derived following single-cell sorting. ⁶⁷ It was seen that leptin caused senescence in the progenitors via the p53/p21 pathway and increased terminal differentiation.

Table 3.

Summary of Chondroprogenitors Derived Following Cell Sorting.

S. No.	Method of Isolation	Cartilage: Species	Isolation Culture Condition			Objectives	Outcome Measures	Results
	Single Cell Sorting		Cartilage Slices: Dimensions	Culture Medium and Additives	Time for Cellular Dissociation/Migration			ISCT Criteria	Reported CD Markers	Differentiation Potential	Overall
1	Yu et al., 2013 66	Bovine femoral condyle: stifle joint	6 mm full thickness biopsy punch: further minced	DMEM + Ham’s F12 (1:1 mixture) + 10% KOSR, 50 µg/µL L-ascorbate	Collagenase type I + pronase (0.25 mg/mL each) in shaking incubator overnight	CPs used as proof of concept: to assess effect of bioprinting on cell phenotype	Cell viability assay, cell functionality by PCR	N	—	—	Model proposed generates vessel-like channels; cells are affected by density used, alginate concentration, dispensing pressure and nozzle size
2	Yu et al., 2014 12	Bovine femoral condyle: stifle joint	6 mm full thickness biopsy punch: further minced	DMEM + Ham’s F12 (1:1 mixture) + 10% KOSR, 50 µg/µL L-ascorbate	Collagenase type I + pronase (0.25 mg/mL each) in shaking incubator overnight	Identification and characterization of clonal populations of single cell sorted cartilage progenitors	Clonogenicity, chemotactic assay, PCR, trilineage, immunostaining: ABCG2 Notch-1 Lubricin	N	—	Adipogenic, osteogenic, chondrogenic	Identification of high efficiency colony forming cells: derivative of single cell cloned progenitor
3	Su et al., 2015 11	Human OA knee joints	<1 mm3	DMEM + 10% FBS	0.2% type II collagenase: 8 hours in shaking water bath at 37°C	Evaluate specificity of CD146 as a marker of chondroprogenitors	FACS, CFU assay, trilineage, PCR	N	CD34, CD144, CD45, CD73, CD90, CD146, CD105, CD166, CD29 , CD31, CD HLA-ABC, CD HLA-DR	Adipogenic, osteogenic, chondrogenic	Due to high migration and survival, CD146+ chondroprogenitors may be tissue specific for cartilage repair
4	Zhao et al., 2016 67	Sprague-Dawley rats joints	<1 mm3	DMEM + 10% FBS + 4.5 mM L-glutamine	0.2% type II collagenase: 8 hours in shaking water bath at 37°C	FACS, immune-cytochemistry, immune-histochemistry, cell migration, trilineage differentiation, cell cycle analysis, senescence assay, cell signaling studies and western blot for phospho-p38 and p21	Evaluate effect of leptin on differentiation and proliferation in CPs to understand roles played by leptin in etiology and development of OA	N	CD90, CD29, CD45, CD49e	Adipogenic, osteogenic, chondrogenic	Link between obesity and cartilage damage induced by leptin-mediated effects on cell behavior, senescence (p53/p21 pathway) and intracellular signaling; leptin decreased chondrogenesis and increased osteogenesis

OA = osteoarthritis, CP = chondroprogenitor, MSC = multipotent mesenchymal stromal cells/mesenchymal stem cells; FBS = fetal bovine serum; KOSR = knockout serum replacement; IU = international unit; DMEM/F12 = Dulbecco’s minimum essential medium/F-12 Ham; FACS = fluorescence-assisted cell sorting; p = passage; CD = cluster of differentiation; CFU = colony forming unit; ABCG2 = AB cassette transporter gene; PCR = polymerase chain reaction.

Discussion

In the field of cartilage repair, cell-based therapy primarily involves MSCs and chondrocytes as regenerative interventions. Due to effective but suboptimal healing observed with their use, search for a cellular substitute led to work on chondroprogenitors. Since the nomenclature for this cell type is diverse, this review was restricted to studies focusing on hyaline cartilage–derived progenitors and their properties. An assessment of data summary led to categorization of all studies into 3 groups based on technique of progenitor isolation.

With regard to growth kinetics, though all chondroprogenitor subgroups displayed clonal growth, migratory potential was consistently assessed and demonstrated in reports using cartilage explant derived progenitors, given that this parameter was a prerequisite to obtain this cell type ( Table 2 ). In contrast, little data are available regarding fibronectin derived chondroprogenitors’ inherent ability to migrate, with studies only examining the influence of chemokines on efficiency of migration. ³⁵ When cell surface markers were assessed, all chondroprogenitor subgroups exhibited high positive and minimal negative MSC marker expression. In terms of a discrete marker, a study reported significantly high levels of CD49b in migratory chondroprogenitors as compared to BM-MSCs, on evaluation of cell surfaceome. ⁵⁷ However, no studies reported notably high expression of any specific marker in fibronectin chondroprogenitors when compared to other cell types, with 2 studies showing similar marker profile between fibronectin progenitors and cultured chondrocytes.^24,27 The challenge with comparing progenitors and chondrocytes lies in that, they are derived from the same source and may inherently exhibit similar phenotypic profiles. Any minor differences that may exist would be altered or lost upon their release from their microenvironment or due to expansion in culture.^24,25,68

On further assessment of chondroprogenitor subgroups, another area of potential conflict could be with regard to culture conditions required for their propagation. There is an apparent species variability wherein progenitors isolated from animal sources do not require additional growth factors in the concentration, as reported for the human subtype. Furthermore, concerning human cells, reports on migratory chondroprogenitors do not employ superadded growth factors, whereas adhesion assay derived progenitors are commonly grown with additives such as TGFβ, FGF, among others ( Tables 1 and 2 ). This observation favors explant derived progenitors over fibronectin assay-derived cells as the use of growth factors to sustain proliferation in culture may also result in additional changes to cell biology, as many additives in common use are important cell signaling molecules.

Although multiple studies have assessed influence of varying culture conditions and interventions on fibronectin progenitor chondrogenesis in vitro, data pertaining to explant derived chondroprogenitors is limited. However, a noteworthy finding reported in studies on both subgroups, was the beneficial effect of PRP or platelet lysate on chondroprogenitor phenotype, as either a biocompatible scaffold or an additive.^17,41,65 Use of autologous/allogeneic platelet products may be investigated in the future as xeno free alternative for chondroprogenitor proliferation and differentiation. With respect to in vivo studies involving chondroprogenitors, fibronectin derived cells demonstrated results superior to MSCs but equivalent or inferior results to chondrocytes in other reports.^1,33 Fibronectin progenitors embedded in scaffolds also, did not exhibit improved healing over the use of plain scaffold. ⁴⁹ Although a single report showed that migratory progenitors encapsulated in PRP displayed better results than scaffolded BM-MSCs and chondrocytes, ⁶⁵ lack of other in vivo data makes it difficult to infer superiority conclusively.

Since multiple studies have demonstrated that hyaline cartilage–derived chondroprogenitors display properties of inherent chondrogenesis and minimal propensity for terminal differentiation, the future direction with translational research involving progenitors should include enhancement of their chondrogenic potential while maintaining their lower hypertrophic preponderance. Following data analysis, it is evident that chondroprogenitors display characteristics akin to MSCs and are a contender for cell-based therapy in cartilage repair. The complications involved in their use stem from the lacunae still present in their biological profile and the myriad ways of isolation and expansion. In particular, no literature was available that elaborated on immunogenic and immunomodulatory properties of chondroprogenitors, which is essential with respect to predicting in vivo behavior of the cell following transplantation. With regard to progenitor subgroups, current evidence points to improved outcomes with the use of migratory progenitors, although a direct comparison between the two cell populations is warranted. Any inferences made from comparisons done on the collated studies also suffer due to the ambiguity in results seen with fibronectin chondroprogenitors, arising due to the culture conditions established for their use and also secondary to limited sample size.

In conclusion, this review reiterates that chondroprogenitors can serve as a useful tool in cell-based therapy for cartilage repair after appropriate optimization. Understanding and further clarification of nomenclature, isolation methodology, along with refinement of culture techniques can yield true chondroprogenitors having multiple applications in the treatment of osteochondral afflictions.